Introduction
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most common cause of cancer-related death worldwide, and patients are being diagnosed at a younger age and more advanced stage, although its annual incidence and mortality rate continue to decline [
1]. Oxaliplatin is currently one of the most effective chemotherapeutic drugs for CRC treatment, and oxaliplatin-based combination chemotherapy regimens, such as FOLFOX, CapeOX and FOLFOXIRI, are widely used as first-line treatments in the clinic [
2]. However, accumulating research has indicated that 40 – 50% of CRC patients will develop drug resistance before or during oxaliplatin treatment, which leads to therapeutic failure and poor prognosis [
3]. The mechanisms of oxaliplatin resistance, a multifactorial phenomenon, have only been partially described [
4]. Therefore, understanding the mechanisms of oxaliplatin resistance and seeking effective strategies to reverse resistance are critical to overcome such challenges and improve patient survival.
Metabolic reprogramming, a hallmark of cancer, is a key contributor to tumour progression and therapeutic resistance [
5]. Metabolic rate-limiting enzymes, which regulate the direction and speed of metabolic pathways, play critical roles in metabolic reprogramming [
6] Strategies that target aberrant metabolism and key metabolic enzymes have great potential as therapeutic options for enhancing the drug susceptibility of cancers [
7]. Inosine 5'-monophosphate dehydrogenase (IMPDH) is one of key rate-limiting enzymes in purine metabolism and plays significant biological functions in normal physiological activities and abnormal diseases [
8]. As one of two distinct isoforms, IMPDH1 is generally less expressed than IMPDH2 is in most tissues [
9] and is the main constitutively expressed protein in normal human leukocytes and lymphocytes [
10]. However, IMPDH2 is particularly overexpressed in rapidly proliferating and neoplastic cells [
11]. Numerous studies have shown that high IMPDH2 expression is associated with tumorigenesis and progression in most cancers [
12,
13], indicating that IMPDH2 may be a significant biomarker [
14]. Inhibition of IMPDH2 activity by mycophenolic acid (MPA) suppressed guanosine triphosphate (GTP) synthesis and increased the radiosensitivity of glioblastoma cells by impairing DNA repair [
15]. However, whether aberrant cancer metabolism supports oxaliplatin resistance remains unknown.
The Wnt/β-catenin signalling pathway plays vital roles in normal embryonic maintenance and development [
16] and can regulate cell proliferation and apoptosis in multiple tumours [
17]. Overactive Wnt signalling leads to decreased degradation of β-catenin and increased entry into the nucleus, which, via lymphoid enhancer factor/T-cell factor (LEF/TCF) transcription factors, results in inappropriate activation of downstream target genes, including c-Myc, Cyclin D1, MMP7, etc. [
18]. Previous studies suggested that aberrant Wnt/β-catenin signalling pathway is involved mainly in cancer cell proliferation and tumour progression [
19], mainly of gastrointestinal origin [
20]. A growing body of research now revealed that upregulated Wnt/β-catenin promotes resistance to chemotherapy in multiple cancers [
21,
22]. The Wnt/β-catenin pathway can direct glycolysis and blocking Wnt/β-catenin signalling reduces glycolytic metabolism and results in the inhibition of tumour growth and angiogenesis [
23,
24]. To date, however, whether the Wnt/β‑catenin pathway affects cancer metabolism and modulates chemoresistance in CRC has not been determined.
In the present study, we investigated the metabolic changes that occur in oxaliplatin-resistant CRC cells and identified increased purine metabolism, which mainly arises from the upregulation of IMPDH2 expression through the Wnt/β‑catenin pathway. IMPDH2 affects resistance to apoptosis induced by oxaliplatin through the Caspase 7/8/9 and PARP1 proteins. These findings suggest that the regulation of purine metabolism by IMPDH2 plays an essential role in oxaliplatin resistance and provide evidence that a novel therapeutic strategy targets IMPDH2 to restore the sensitivity of CRC to oxaliplatin.
Materials and methods
Drugs and chemicals
Oxaliplatin (S1224) was purchased from Selleck. Guanylic acid disodium salt (GMP, GC35159) was purchased from GlpBio. Mycophenolic acid (MPA, HY-B0421), Mycophenolate mofetil (MMF, HY-B0199) and XAV939 (HY-15147) were obtained from MedChemExpress.
Cell lines and culture
The human CRC cell lines SW620, RKO, HCT116 and HCT8 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HEK293T cells were obtained from the ATCC (USA). Oxaliplatin-resistant HCT8/L-OHP cells were established by continuously culturing parental HCT8 cells in the presence of stepwise increasing concentrations of oxaliplatin over approximately 10 months. SW620, HCT116, HCT8 and HCT8/L-OHP cells were cultured in RPMI-1640 medium (Gibco, USA), while RKO and HEK293T cells were grown in high-glucose DMEM (Gibco, USA). All culture media contained 10% foetal bovine serum (Hyclone, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, USA). All cell lines were maintained in a humidified chamber with 5% CO2 at 37 ℃.
Establishment of oxaliplatin-resistant cells
To establish oxaliplatin-resistant cells, parental HCT8 cells were initially cultured in RPMI-1640 medium supplemented with 1 µmol/L oxaliplatin for 48 h. The drug concentration was increased by 2 µmol/L once the cells recovered to normal growth after 2 weeks of continuous drug exposure. The 50% inhibitory concentration (IC50) of oxaliplatin at each dose was determined by a CCK-8 assay, and the resistance index (RI) of the oxaliplatin-resistant cells was defined as follows: RI = IC50 of resistant cells/IC50 of parental cells. Stable oxaliplatin-resistant cells were selected and maintained at a final drug concentration of 25 µmol/L after approximately 10 months, and the induced oxaliplatin-resistant cells were named HCT8/L-OHP. Single cell-derived clones of HCT8/L-OHP cells were obtained by a limiting dilution strategy and were used for further study. For all experiment, the HCT8/L-OHP cells were refreshed with oxaliplatin-free medium for 48 h before treatment.
Flow cytometry analysis of cell apoptosis
Cells were seeded at 2 × 105 cells per well into 12-well plates with three duplicate wells. After 24 h of incubation, the cells were treated with different concentrations of oxaliplatin for another 24 h. Subsequently, the cells were washed with PBS, harvested using trypsin without EDTA, resuspended in binding buffer and stained with Annexin V-APC/PI (Multi Sciences, AP107) for 15 min in the dark according to the manufacturer's protocols. The percentage of apoptotic cells was measured using a CytoFLEX flow cytometer (Beckman Coulter) and analysed using CytExpert software (Beckman Coulter). The results are presented as the percentage of total cells which were living cells, early apoptotic cells, late apoptotic cells and necrotic cells.
Overexpression and knockdown of IMPDH2 in CRC cells
For ectopic overexpression of IMPDH2, the coding sequence of Flag-tagged IMPDH2 was amplified via PCR and inserted into the pcDNA3.1 (+) vector. HCT8 and SW620 cells were transiently transfected using Lipofectamine™ 3000 (Invitrogen, L3000015) according to the manufacturer's instructions. After 24 or 48 h of transfection, the following series of experiments were performed.
To establish stable IMPDH2-knockdown cells, the short hairpin RNA (shRNA) for IMPDH2 was cloned and inserted into the pLKO.1-puromycin lentiviral vector. Then HEK293T cells were cotransfected with the psPAX2 packaging plasmid and pMD2.G envelope plasmid using Lipofectamine™ 3000 to produce lentivirus. After transfection for 48 h, the viral supernatants were collected and used to infect 5 × 105 HCT8/L-OHP cells after filtration, followed by selection with 2 µg/mL puromycin selection. The IMPDH2 knockdown efficiency was verified by qPCR and western blotting analysis. The sequences of IMPDH2-shRNA were as follows: IMPDH2-shRNA-F: 5ʹ-CCGGCGGAAAGTGAAGAAATATGAACTCGAGTTCATATTTCTTCACTTTCCGTTTTTG-3ʹ.
CCK-8 assay
For the CCK-8 assay, 6 × 103 cells in 100 µL culture medium were seeded in a 96-well plate with three duplicate wells and incubated for 24 h. Then the cells were treated with various concentrations of oxaliplatin. After 48 h of treatment, 10 µL of the CCK-8 reagent (A311-02) was added to each well and the 96-well plate was subsequently incubated at 37 ℃ for 2 or 4 h. The optical density (OD) value at 450 nm was assessed to determine the inhibition ratio of the cells with an EPOCH spectrophotometer (BioTek Instruments). All the experiments were performed with at least three replicates.
Total RNA was extracted from cells using TRIzol (Invitrogen, 15596018) according to the manufacturer’s instructions. The extracted total RNA was measured on a NanoDrop spectrophotometer (NanoDrop Technologies, USA) and 500 ng of RNA was reverse transcribed to cDNA using a reverse transcription kit (Takara, RR047A). QPCR was performed using SYBR Green reagent (Takara TB Green, RR420A), and the mRNA level was detected by an ABI Step-One Detection System. Each sample was analysed in triplicate. The relative mRNA expression levels were measured using the 2
−ΔΔCt method and β-actin was used as the internal reference. Primers sequence of qPCR were listed in Table
1.
Table 1
Primers Sequence of qPCR used in this study
PRPS1 | Forward: 5ʹ-ATCTTCTCCGGTCCTGCTATT-3′ |
| Reverse: 5ʹ-TGGTGACTACTACTGCCTCAAA-3ʹ |
PRPS2 | Forward: 5ʹ -AGCTCGCATCAGGACCTGT-3ʹ |
| Reverse: 5ʹ-ACGCTTTCACCAATCTCCACG-3ʹ |
PPAT | Forward: 5ʹ-GATGGGAGTTCGGTGCCAA-3ʹ |
| Reverse: 5ʹ-CAACGAAGGGCTGACAATTTTC-3ʹ |
IMPDH1 | Forward: 5ʹ-TGAAGAAGAACCGAGACTACCC-3ʹ |
| Reverse: 5ʹ-TCCAGACGGTATTTGTCATCCT-3ʹ |
IMPDH2 | Forward: 5ʹ-AGGGAAAGTTGCCCATTGTAAA-3ʹ |
| Reverse: 5ʹ-TGGGTAGTCCCGATTCTTCTTC-3ʹ |
GMPS | Forward: 5ʹ-ATGGCTCTGTGCAACGGAG-3ʹ |
| Reverse: 5ʹ-CCTCACTCTTCGGTCTATGACT-3ʹ |
ADSS1 | Forward: 5ʹ-AAGAAGGGAATCGGACCAACC-3ʹ |
| Reverse: 5ʹ-CCGTGGAGTGCCTCATACATAA-3ʹ |
ADSS2 | Forward: 5ʹ-TGGGTATGCCACCTCAAAATG-3ʹ |
| Reverse: 5ʹ-GCTCTGTAGGAAAGGCACCAATA-3ʹ |
ADSL | Forward: 5ʹ-GCTGGAGGCGATCATGGTTC-3ʹ |
| Reverse: 5ʹ-TGATAGGCAAACCCAATGTCTG-3ʹ |
APRT | Forward: 5ʹ-GGCCGCATCGACTACATCG-3ʹ |
| Reverse: 5ʹ-CTCAGCCTTCCCGTACTCC-3ʹ |
HPRT1 | Forward: 5ʹ-ACCAGTCAACAGGGGACATAA-3ʹ |
| Reverse: 5ʹ-CTTCGTGGGGTCCTTTTCACC-3′ |
ADAR | Forward: 5ʹ-CTGAGACCAAAAGAAACGCAGA-3ʹ |
| Reverse: 5ʹ-GCCATTGTAATGAACAGGTGGTT-3′ |
β-catenin | Forward: 5′-AAAGCGGCTGTTAGTCACTGG-3′ |
| Reverse: 5ʹ-CGAGTCATTGCATACTGTCCAT-3ʹ |
c-Myc | Forward: 5ʹ-GGCTCCTGGCAAAAGGTCA-3ʹ |
| Reverse: 5ʹ-CTGCGTAGTTGTGCTGATGT-3ʹ |
Cyclin D1 | Forward: 5ʹ-GAACACGGCTCACGCTTAC-3ʹ |
| Reverse: 5ʹ-CCCAGACCCTCAGACTTGC-3ʹ |
β-actin | Forward: 5ʹ-CATGTACGTTGCTATCCAGGC-3ʹ |
| Reverse: 5ʹ-CTCCTTAATGTCACGCACGAT-3ʹ |
Western blotting analysis
Cell lysates for western blotting were collected in RIPA lysis buffer containing protease inhibitor cocktail (Thermo, 78,443) for 30 min on ice and centrifuged at 12,000 × g for 15 min at 4 ℃ to obtain the supernatant. Protein quantification was performed with the Bradford Protein Assay Kit (Thermo, 23,236). Proteins were separated by SDS‒PAGE electrophoresis and subsequently transferred to PVDF membranes (Millipore, ISEQ00010). Then the membranes were blocked with 5% skim milk (BD Difco, 232,100) in Tris-buffered saline containing 0.5% Tween-20 for 1 h and incubated with primary antibodies at 4 ℃ overnight. The primary antibodies included IMPDH2 (1:5000 dilution, #ab131158, Abcam), Caspase 3/p17/p19 (1:3000 dilution, #66470-2-Ig, Proteintech), Caspase 7/p20 (1:1000 dilution, #27155-1-AP, Proteintech), Caspase 8/p43/p18 (1:5000 dilution, #66093-1-Ig, Proteintech), Caspase 9/p35/p10 (1:2000 dilution, #66169-1-Ig, Proteintech), Bax (1:10000 dilution, #60267-1-Ig, Proteintech), PARP1 (1:20000 dilution, #66520-1-Ig, Proteintech), β-Catenin (1:1000 dilution, #8480S, Cell Signaling Technology), Cyclin D1 (1:1000 dilution, #2978S, Cell Signaling Technology), C-Myc (1:1000 dilution, #ab32072, Abcam), P-gp (1:1000 dilution, #22336-1-AP, Proteintech), β-actin (1:5000 dilution, #20536-1-AP, Proteintech). After incubation with the appropriate secondary antibodies (anti-rabbit IgG, 1:10000 dilution, #A16098, Thermo; anti-mouse IgG, 1:10000 dilution, #PA1-28555, Thermo). The protein signals were visualized by an enhanced chemiluminescence (ECL) detection kit (NCM Biotech, P10300).
Untargeted metabolomics analysis was conducted by the liquid chromatography-tandem mass spectrometry (LC–MS/MS) technology, using a Waters 2D UPLC (Waters, USA) coupled with a high-resolution mass spectrometer Q Exactive HF (Thermo Fisher Scientific, USA) with a heated electrospray ionization (HESI) source and controlled by the Xcalibur 2.3 software program (Thermo Fisher Scientific, Waltham, MA, USA). Five microlitres of each sample was injected onto a Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters, USA) for chromatographic separation, and the column temperature was maintained at 45 ℃. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B) in positive mode, and in negative mode, the mobile phase consisted of 10 mM ammonium formate (A) and acetonitrile (B). The gradient conditions were as follows: 0–1 min, 2% B; 1–9 min, 2%-98% B; 9–12 min, 98% B; 12–12.1 min, 98% B to 2% B; and 12.1–15 min, 2% B. The flow rate was 0.35 mL/min. Data processing was performed using the Compound Discoverer 3.1 (Thermo Fisher Scientific, USA) software. The mass spectrometric settings for positive/negative ionization modes were as follows: spray voltage, 3.8/ − 3.2 kV; sheath gas flow rate, 40 arbitrary units (arb); aux gas flow rate, 10 arb; aux gas heater temperature, 350 ℃; and capillary temperature, 320 ℃. The full scan range was 70–1050 m/z with a resolution of 70,000, and the automatic gain control (AGC) target for MS acquisitions was set to 3e6 with a maximum ion injection time of 100 ms. The top 3 precursors were selected for subsequent MSMS fragmentation with a maximum ion injection time of 50 ms and resolution of 30,000; the AGC was 1e5. The stepped normalized collision energy was set to 20, 40 and 60 eV. The samples were randomized to reduce systematic errors. A quality control (QC) sample containing equal aliquots of all cell samples was interspersed for every 10 samples.
Immunoprecipitation-mass spectrometry (IP-MS) analysis
LC‒MS/MS was used to identify the differentially expressed proteins in HCT8 and HCT8/L-OHP cells by transfecting cells with Flag-tagged IMPDH2 plasmids. Briefly, total cell lysates from HCT8 and HCT8/L-OHP cells were collected and incubated with anti-DDDDK-tag mAb-magnetic beads (MBL, M185-11R) at 4 ℃ overnight. Then, the beads were separated by a magnetic device. The cell proteins were identified via LC‒MS/MS using a Q Exactive HF X mass spectrometer (Thermo Fisher Scientific, USA). Subsequently, functional annotation analysis and pathway analysis were performed on the final protein identification list.
Animal experiment
All animal experiments were conducted in accordance with the Animal Ethics Committee of The Sixth Affiliated Hospital, Sun Yat-sen University (IACUC-2023051101) and all relevant ethical regulations were strictly followed. Male BALB/c nude mice aged 4–6 weeks were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd (Zhejiang, China). Approximately 5 × 106 HCT8/L-OHP cells were subcutaneously injected into the right flank in 100 μL of 50% PBS and 50% Matrigel-basement membrane matrix. Once the tumour volume reached 100 mm3, the mice were randomly divided into four experimental groups: control, oxaliplatin alone, MMF alone, or a combination of oxaliplatin and MMF. Oxaliplatin was intraperitoneally injected at a dose of 10 mg/kg every 3 days, and MMF was intragastrically administered at a dose of 120 mg/kg twice a day. The tumour volume and body weight were measured every 3 days. The tumour volume was calculated by the formula: Volume = length × width2 × 0.5 cm3.
Statistical analysis
All the data are presented as the means ± SDs of at least three independent experiments. The statistical analysis was performed using GraphPad Prism 8 software (San Diego, 265 California), and the statistically significant differences were analysed using two-tailed Student’s t tests. The statistical significance was defined as follows: ns = not significant, *p < 0.05, **p < 0.01 and ***p < 0.001; these data were included in the respective figure legends.
Discussion
Chemoresistance remains a major challenge in the management of patients with CRC. Several mechanisms of oxaliplatin resistance have been reported, including reduced cellular drug uptake and accumulation [
26], increased repair of DNA damage [
27], induction of epithelial-mesenchymal transition (EMT) [
28], and desensitization to cell apoptosis [
29]; however, effective strategies for reversing resistance have not been achieved. Emerging evidence suggests that aberrant cancer metabolism could impart drug resistance [
30]. In the present study, we characterized the role of metabolic rewiring in oxaliplatin resistance and identified that purine metabolism strongly correlates with oxaliplatin resistance in CRC. The Wnt/β-catenin pathway activates IMPDH2-mediated purine metabolism to reduce oxaliplatin-induced apoptosis in CRC. We also demonstrated that knockdown and pharmacological inhibition of IMPDH2 could reverse oxaliplatin resistance in vivo and in vitro.
Aberrant cancer metabolism is considered one of the important hallmarks of cancer cells [
31]. Generally, rapidly proliferating cancer cells require both elevated energy consumption and increased nucleotide biosynthetic pathways to sustain high levels of DNA replication and RNA transcription [
32]. Purine metabolism is the most important source of nucleotide biosynthesis, and increased purine metabolites can accelerate cancer cell proliferation and tumour progression [
33]. Targeting IMPDH, a key regulator of purine metabolism, effectively suppressed hepatocellular carcinoma progression [
34]. Another study showed that purine metabolites, especially guanylates, strongly correlate with radiation resistance in glioblastoma and that inhibiting GTP synthesis radiosensitizes glioblastoma cells [
15]. These studies imply that inhibiting key rate-limiting enzymes or reducing intermediate metabolites of purine metabolism may be attractive strategies for cancer therapy. Consistently, using an untargeted metabolomics approach, we identified that the metabolic levels of purine metabolism products, especially GMP, a vital intermediate metabolite, were significantly elevated in oxaliplatin-resistant cells. We further confirmed that the sensitivity of cells to oxaliplatin decreased when GMP was exogenously supplemented in oxaliplatin-sensitive CRC cells. However, the inhibition of GMP synthesis by MPA or MMF, which are currently available clinical therapeutic drugs, impaired oxaliplatin resistance, led to significant cell death in CRC cells in vitro, and even suppressed xenograft tumour growth in nude mice.
Interestingly, oxaliplatin cytotoxicity has been primarily ascribed to interactions between oxaliplatin and DNA that can form DNA adducts that lead to cell cycle arrest and apoptosis [
35]. Mechanistically, purine metabolites provide an important substrate pool for DNA replication and RNA transcription to fuel the biosynthetic demands of cell growth and division. Activated purine metabolism, especially GMP, can reverse DNA damage caused by oxaliplatin, thus contributing to resistance in CRC. IMPDH is one of the major checkpoints for the activity of purine metabolism; this enzyme catalyses the transformation of inosine monophosphate (IMP) to XMP, which is further converted into GTP for DNA and RNA synthesis [
14]. Inhibition of IMPDH results in decreased cellular guanine nucleotide pools and subsequently suppresses the synthesis of DNA and RNA, thus inhibiting cancer cell proliferation by blocking cell cycle progression and inducing cell apoptosis [
36]. Accumulating evidence suggests that IMPDH2 is the predominant isoform of IMPDH, and increased IMPDH2 expression is associated with tumorigenesis, metastasis and recurrence in most cancers, including leukaemia [
37], prostate cancer [
38], ovarian cancer [
39], non-small cell lung cancer [
40], and triple-negative breast cancer [
41]. In particular, Duan et al. reported that IMPDH2 is highly expressed in CRC and is correlated with poor survival in CRC patients, and the overexpression of IMPDH2 dramatically promoted CRC progression [
13]. Herein, we found that both IMPDH1 and IMPDH2 were significantly upregulated in oxaliplatin-resistant CRC cells, whereas the increase in IMPDH2 was twofold greater than that in IMPDH1. Further studies revealed that CRC cells with higher IMPDH2 expression were more resistant to oxaliplatin-induced apoptosis. Overexpression of IMPDH2 in CRC cells resulted in reduced cell death upon treatment with oxaliplatin, whereas knockdown of IMPDH2 led to increased sensitivity to oxaliplatin. Western blotting analysis demonstrated that IMPDH2 inhibited cell apoptosis through the prevention of the accumulation of cleaved caspase 7, caspase 8, caspase 9, and PARP1, thereby promoting resistance to oxaliplatin in CRC cells. Moreover, the combination of the IMPDH2 inhibitor MPA or the addition of the GMP substrate with oxaliplatin produced similar results. Thus, we hypothesize that purine reprogramming is a novel mechanism of oxaliplatin resistance and that inhibiting purine metabolism by targeting the key enzyme IMPDH2 may be a potential therapeutic approach for reversing oxaliplatin resistance in CRC.
The Wnt/β-catenin signalling pathway not only plays an essential role in tumorigenesis and cancer progression [
17], but is also associated with chemotherapeutic resistance in multiple cancers [
21,
42]. Additionally, aberrant activation of Wnt/β-catenin signalling could not only directly induce glucose metabolic reprogramming and promote colon cancer cell proliferation [
23] but also indirectly activate downstream transcription factors such as HIF-1ɑ to induce metabolic reprogramming and impart 5-fluorouracil resistance in CRC [
42]. In the present study, we discovered that the Wnt/β‑catenin signalling pathway is hyperactivated in oxaliplatin-resistant CRC cells. The activated Wnt/β‑catenin signalling pathway promote IMPDH2 expression, whose increase, in turn, induced the expression of β‑catenin, forming a Wnt/β-catenin/IMPDH2 positive feedback circuit that confers resistance to oxaliplatin in CRC. We further performed flow cytometry analysis, and the results showed that the combination of XAV939, a tankyrase inhibitor that targets Wnt/β-catenin, with oxaliplatin markedly increased cell apoptosis when compared with XAV939 or oxaliplatin alone; however, these effects were nearly reversed by the addition of GMP. Taken together, our study revealed that the Wnt/β‑catenin signalling pathway may be a master regulator of purine metabolic reprogramming in oxaliplatin resistance. However, c-Myc also serves as an oncogene that directly induces enhanced expression of target enzymes involved in the metabolism of a variety of enzymes, including IMPDH2 [
43]. Thus, the mechanism underlying the interaction between c-Myc and the Wnt/β‑catenin pathway and IMPDH2 in CRC oxaliplatin resistance remains to be assessed in our future research. Additionally, tumour-infiltrating immune cells (TIICs) play significant roles in chemotherapeutic resistance in rectal cancer [
44]. MMF has been widely approved as an immunosuppressant in organ transplant recipients and can also mediate the tumour microenvironment [
45]. Therefore, it is reasonable to assume that MMF, or targeting IMPDH2, may exert anticancer activity via its immunosuppressive effects on TIICs, but further studies are needed.
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