Background
Colorectal cancer (CRC) is one of the most common types of malignancies worldwide [
1], and its incidence and mortality rates are continuously increasing. Despite the fact that improvements have been made in diagnostic methods and therapeutic strategies, the overall prognosis of CRC patients still remains pessimistic. Hence, it is desperately needed to improve our identification of the molecular mechanisms underlying CRC progression and to develop more efficient therapeutic methods of managing CRC.
Inosine5′-monophosphate dehydrogenase (IMPDH) is a rate-limiting
enzyme which catalyzes the
nicotinamide adenine dinucleotide (NAD
+)-dependent oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), which is an essential step in de novo biosynthesis of guanine nucleotides [
2]. IMPDH is a key regulator of the intracelluar guanine nucleotide pool, demonstrating its importance for DNA and RNA synthesis. Human IMPDH is a tetramer composed of approximately 55 kDa monomers [
3] and has two distinct isoforms, IMPDH1 and IMPDH2, with an 84% similarity in their amino acid sequence [
4]. IMPDH1 is generally expressed in normal human leukocytes and lymphocytes, whereas IMPDH2 is generally upregulated in tumor tissues and proliferating cells [
5‐
7]. Most importantly, the increase in total IMPDH activity is mainly attributed to increased expression of IMPDH2 [
4]. Nowadays, isoforms of IMPDH, particularly IMPDH2, have been of particular interest to oncologists due to its roles in regulation of cell proliferation, cell differentiation, and chemoresistance [
4,
8‐
11].
Accumulating evidence reveals that IMPDH2 was significantly elevated in multiple types of tumor cells and associated with cancer progression and poor prognosis of tumor patients [
12‐
14]. For instance, increased IMPDH2 expression was observed in human melanoma cell lines [
15], human ovarian tumors [
13], human leukemic cell lines [
7] and multiple myeloma cells [
16]. A study by Fellenberg et al. showed that IMPDH2 could be served as a promising candidate for the stratification of osteosarcoma patients into low- and high-risk groups [
8]. Furthermore, inhibition of IMPDH2 activity increased sensitivity to methotrexate in HT29 human colon cancer cells [
17], and induced growth arrest of human multiple myeloma cells [
16]. To date, however, the biological role of IMPDH2 in CRC progression and its molecular mechanisms have not been well elucidated.
In our study, IMPDH2 was shown to be highly expressed in CRC cell lines and tissues. A series of in vitro and in vivo assays revealed that overexpressing IMPDH2 dramatically promoted the proliferation, invasion and migration, tumorigenicity and epithelial–mesenchymal transition (EMT) of CRC cells, while knockdown of IMPDH2 had the opposite effect. We further demonstrated that IMPDH2 overexpression accelerated G1/S phase cell cycle transition by inducing increased expression of cyclin D1 and Ki-67 and downregulation of p21Cip1 and p27Kip1. More importantly, G1/S phase cell cycle transition was triggered by IMPDH2 through activation of AKT activity, downregulation of mTOR and FOXO1 transcriptional activity. Additionally, inhibition of the mTOR pathway could induce suppression of invasion, migration and EMT in IMPDH2-overexpressed cells. These findings suggest that IMPDH2 plays a potential oncogenic role in CRC progression and represents a promising prognostic marker of this disease.
Methods
Cell culture
Human embryonic kidney 293 T cells, normal human colon epithelial cells (FHC (CRL-1831)) and seven human CRC cell lines, including HCT116, SW620, M5, SW480, HT29, DLD-1 and LoVo were obtained from a cell bank at the Chinese Academy of Sciences (Shanghai, China). All cells were authenticated by short tandem repeat (STR) profiling after receipt and were propagated for less than 6 months after resuscitation. All CRC cell lines were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) with 10% fetal bovine serum (HyClone, Logan, USA) and 100 U/ml penicillin/ streptomycin (Gibco). These cell lines were maintained in a humidified chamber containing 5% CO2 at 37 °C.
Tissue preparation
For western blotting and quantitative real-time PCR (qPCR) analyses, 34 pairs of fresh CRC tissues and matched adjacent normal colorectal tissues from primary CRC patients were obtained in operation from Nanfang Hospital, Southern Medical University (Guangzhou, China). Paraffin-embedded specimens of 214 primary CRC patients who undergone elective operation were collected from Nanfang Hospital between February 2009 and June 2011. None of these patients received any preoperative chemotherapy or radiotherapy. The stage of disease was determined according to the tumor size, lymph node involvement and distant metastasis (pTNM) classification system [
18]. Complete follow-up, ranging from 1 to 96 months, was available for the cohort of 214 patients, and the median survival was 53 months. The study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University and all aspects of the study comply with the Declaration of Helsinki. Written informed consent was obtained from all patients.
Immunohistochemistry
The expression level of IMPDH2 protein in 214 pairs of paraffin-embedded CRC tissues and matched adjacent normal colorectal tissues was examined by immunohistochemistry (IHC). The sections were heated, deparaffinized, rehydrated and placed insodium citrate buffer (pH = 6.0) for antigen retrieval. Then the slides were immersed in 3% hydrogen peroxide to inhibit the endogenous peroxidase activity. After rinsing three times, the sections were incubated with primary antibody (rabbit anti-IMPDH2, 1:800 dilution, #ab131158; Abcam, Cambridge, UK) overnight at 4 °C, followed by treatment with secondary antibody (anti-rabbit IgG, 1:2000 dilution, #7074; Cell Signaling, Danvers, MA, USA) for 40 min at 37 °C. After being stained with 3,3-diaminobenzidine (DAB), the slides were counterstained with Mayer’s haematoxylin, dehydrated and mounted.
IHC scoring based on the staining intensity and the proportion of positive tumor cells was performed by two independent pathologists blinded to the clinical data. The staining intensity was scored as 0 (negative), 1 (weak), 2 (medium), 3 (strong). The extent of staining was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%), according to the percentage of the positive staining areas in relation to the whole tumor area or the entire section for the normal sample. The sum of the intensity and extent scores was used as the final staining score (0–7) for IMPDH2. For statistical analysis, a final staining score of ≥3 was considered to be high, and the scores of < 3 as low expression of IMPDH2.
RNA extraction and qPCR
Total RNA from cultured cells and fresh tissues was extracted with Trizol regent (Invitrogen, Calsbad, CA). Synthesis of cDNA was performed by using the PrimeScript RT reagent Kit (Promega, Madison, WI, USA). The SYBR Premix EX Taq™ (Takala, Dalian, China) was used for quantitative real-time PCR (qPCR) operated with an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, USA). The primer sequences used to amplify IMPDH2 were: 5′- GTTTCTGCGGTATCCCAATC -3′ (forward) and 5′- CGAGCAAGTCCAGCCTAT-3′ (reverse). GAPDH was used as an endogenous control. Relative gene expression was determined by the comparative 2-ΔΔCT method.
Western blotting analysis
Proteins from cell and tissue lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Pall Corp, Port Washington, NY). Then the membranes were blocked with 5% skimmed milk and incubated using primary antibodies against IMPDH2 (1:1000 dilution), anti-GAPDH, anti-GSK3β, anti-p-GSK3β, anti-AKT, anti-p-AKT (Ser473), anti-FOXO1, anti-p-FOXO1, anti-mTOR, anti-p-mTOR (Cell signaling Technology, Beverly, MA), E-cadherin (1:1000 dilution,#SAB4503751; Sigma Aldrich), β-catenin (1:1000 dilution, #C2206; Sigma Aldrich), Vimentin (1:1000 dilution, #V6630; Sigma Aldrich), Snail (1:1000 dilution, #SAB1306281; Sigma Aldrich), followed by incubation with the appropriate secondary antibodies (anti-rabbit IgG, 1:3000 dilution, #7074; Cell Signaling). An enhanced chemiluminescence (Pierce, Rockford, IL, USA) was used to detect signals.
Lentiviral transduction and transfection
Overexpression and short hairpin RNA (shRNA) -induced downregulation of IMPDH2 in CRC cells were achieved using the GV367 and GV248 shRNA lentiviral vector (genechem, Shanghai, China). The human shRNA sequences to inhibit IMPDH2 expression are listed as follows: IMPDH2 shRNA: GGACAGACCTGAAGAAGAA (genechem, Shanghai, China). The vectors were packaged in 293 cells. Recombinant lentiviruses were produced by transient transfection of HEK293T cells. Then, transduced cells were selected for 7 days with 0.6 mg/mL puromycin. Protein and mRNA of transfer cells were taken for qPCR and western blotting analyses.
Cell proliferation assay
1 × 103 cells were seeded on 96-well plates and cultured for 24 h.2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfothenyl)-2H-tetrazolium salt (CCK-8, Dojindo, Rockville, USA) solution was added to each well and incubated for 2 h, and then the absorbance of each well was measured at 450 nm with a Microplate Autoreader (Bio-Rad, Hercules, CA, USA). The experiment was performed with three replicates.
Cells were plated on 6-well plates (200 cells/well) and maintained for 2 weeks. The colonies were stained with 1% crystal violet for 30 s after fixation with 4% paraformaldehyde for 30 min. The number of colonies, defined as > 50 cells/colony, was counted. Three independent experiments were performed.
Cell wound healing assay
Cell motility was measured with wound healing assay. 1.2 × 106 Cells were seeded on 6-well tissue culture plates and incubated for 24 h. Scratch wounds were produced by a 10 μL pipette tube, after three washes with cold PBS. And the spread of wound closure was observed after 0 and 48 h, singly. Images were taken to assess the level of migration in each group of transfected cells. Cell motility was quantified by measuring the distance between the advancing margins of cells in three randomly selected microscopic fields (× 200) at each time point.
Transwell migration assay
2 × 105 cells suspended in serum-free media were placed in the upper compartment of 8-μm-pore Transwells (BD Biosciences, San José, CA, USA) and 10% FBS as a chemo-attractant was filled in the lower compartment. The cells were incubated at 37 °C for 2 days. The successfully translocated cells were stained with 0.5% crystal violet for 15 min and calculated in five randomly chosen fields (× 200) under a microscope.
Immunofluorescent assay
Cells were seeded on Confocal plates per well for 48 h and incubated with primary antibodies against E-cadherin (1:200 dilution, #ab1416; Abcam, Cambridge, UK) and Fibronectin (1:200 dilution, #ab2413; Abcam, Cambridge, UK), then followed by incubation with green goat anti rabbit and red goat anti rabbit antibodies. After counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Sigma), an Olympus FV1000 confocal laser-scanning microscope (Olympus America Inc., NY, USA) was used to take images.
Animal experiments
Balb/C-nu/nu athymic nude mice (3–4 weeks old) were obtained from the Laboratory Animal Centre of Southern Medical University, which is certified by the Guangdong Provincial Bureau of Science. To explore the function of the IMPDH2 gene in colorectal tumour growth in vivo, 2 × 106 cells suspended of LoVo with stable overexpression of IMPDH2 and HCT116 with stable knockdown of IMPDH2, or mock cells, were respectively injected into left and right bilateral hind leg subcutaneous of mice. All mice were housed and maintained under specific pathogen-free conditions, and all experiment were approved by the Use Committee for Animal Care and performed in accordance with institutional guidelines. These mices were allocated randomly into each of the four groups (n = 6 per group). Tumour volume was estimated from two perpendicular axes using a digital calipers every two days (volume = (length×width2)/2 and tumour were also photographed. The primary tumour was removed surgically, fixed, paraffin-embedded, and sectioned. The sections were observed under a microscope after haematoxylin and eosin (H&E) staining.
For the in vivo metastasis assay, 2 × 105 of HCT116 cells stably expressing control or IMPDH2 shRNA in a volume of 150 μl in the PBS were injected into nude mice (n = 6 per group) via the tail vein, respectively. Lung metastases of tumor cells were observed 2 months post-injection. The lungs were removed by dissection away from adjacent organs and fixed with 10% formalin. The consecutive tissue sections were obtained and stained with haematoxylin-eosin (H&E) to observe the metastatic nodules of lungs under a microscope. All animal experiments were conducted in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Southern Medical University.
Gene set enrichment analysis (GSEA)
To gain insight into IMPDH2-mediated molecular pathways in colorectal cancer, GSEA was performed using the Broad Institute GSEA version 4.0 software. The Cancer Genome Atlas (TCGA) database consisted of 644 colorectal cancer tissues samples was downloaded from the TCGA (TCGA,
https://cancergenome.nih.gov/). The gene sets used for the enrichment analysis were downloaded from the Molecular Signatures Database (MsigDB,
http://software.broadinstitute.org/gsea/index.jsp). The gene sets with a false discovery rate (FDR) less than 0.25 were considered as significantly enriched.
Statistical analysis
All statistical analyses were carried out using the SPSS 19.0 statistical software package. In at least three independent experiments, the data were presented in terms of the mean ± SD. The Student t-test and the one-way ANOVA test were carried out for qPCR. Comparisons between groups for statistical significance were carried out with a 2-tailed paired Student t-test. The correlation between the expression of IMPDH2 and clinicopathologic factors was evaluated using Pearsonʼs chi-squared (χ2) test. For patients with different levels of IMPDH2 expression, the survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. The Cox proportional hazard model was used for multivariate analysis. P<0.05 was considered statistically significant.
Discussion
In the current study, we highlighted that elevated expression of IMPDH2 was closely associated with several aggressive features and unfavorable prognosis of CRC patients. Overexpression of IMPDH2 could promote the proliferation, invasion, migration and tumorigenicity of CRC cells. Further studies uncovered that IMPDH2 exerted its oncogenic roles by promoting EMT and accelerating the G1/S phase transition in CRC. The above findings provide strong evidences to support the fact that IMPDH2 plays vital roles in the development and progression of CRC and may be a novel therapeutic target.
IMPDH is known as a key rate-limiting enzyme in de novo guanine nucleotide biosynthesis, the inhibitors of which is being widely used in cancer, immunosuppressive and anti-viral research and treatment [
22‐
24]. Inhibition of IMPDH was capable of blocking cell-cycle progression in human T lymphocytes and suppressing the growth of human multiple myeloma cells [
10,
25]. IMPDH2 is believed to be a fascinating target for cancer therapy due to its overexpression particularly in rapidly proliferating and neoplastic cells. A growing number of studies have demonstrated that IMPDH2 was closely implicated in cellular proliferation and tumorigenesis [
4,
26‐
28]. Herein, we found that IMPDH2 was upregulated at the mRNA and protein level in CRC cell lines, in agreement with a previous study [
17]. Then by data-mining in TCGA, we showed that IMPDH2 mRNA was significantly overexpressed in CRC tissues samples. Clinically, elevated expression of IMPDH2 in CRC tissues was further confirmed by qPCR, western blotting and immunohistochemistry analysis. Additionally, the statistical analysis revealed that high IMPDH2 expression significantly correlated with T stage, lymph node state, distant metastasis, lymphovascular invasion and clinical stage and was strongly associated with shorter survival of CRC patients. Moreover, multivariate analysis implied that lymph node state, distant metastasis and IMPDH2 expression could be independent prognostic factors for CRC patients. Our further in vivo and in vitro experiments revealed that IMPDH2 was critically involved in regulation of the proliferation, migration, invasion and tumorigenicity of CRC cells. In the light of these findings, we concluded that IMPDH2 may play oncogenic roles in CRC.
EMT is a key mechanism involved in the complex process of tumor invasion and metastasis. During cancer progression, EMT can alter adhesion of epithelial cancer cells and allow them to invade and migrate to the distant sites, thus contributing to tumor metastasis [
29,
30]. Furthermore, EMT-linked loss of the basement membrane (BM) was closely linked to poor prognosis of CRC patients [
30]. In the current study, we found that overexpressing IMPDH2 decreased the level of E-cadherin and increased that of Vimentin and Snail in CRC cells. On the contrary, silencing of IMPDH2 induced converse results. The results of immunofluorescent assay further revealed that E-cadherin was lowly expressed in the IMPDH2-overexpressed CRC cells while Fibronectin was highly expressed. These data demonstrate that IMPDH2 overexpression can promote the EMT of CRC cells.
Recently, microRNA-34a has been supposed to downregulate the GTP-dependent Ras signaling pathway by targeting IMPDH in different types of malignant cells [
31]. Activation of the tumor suppressor p53 can inhibit cellular IMPDH2 activity and reduce cellular GTP level, thereby repressing cancer cell growth [
32]. Furthermore, Inactivation of IMPDH triggers apoptosis to inhibit the growth of multiple myeloma cells primarily via a caspase-independent, Bax/AIF/Endo G pathway [
16]. There is evidence that IMPDH2 interacts with the pleckstrin homology domain of PKB/AKT in the regulation of GTP biosynthesis [
33]. Herein, we found that the gene set related to cell cycle positively correlated with elevated IMPDH2 expression by GSEA. It is well established that cell-cycle progression is one of the most predominant factors to promote cell proliferation. However, the underlying mechanisms of IMPDH2 involved in cell proliferation of CRC cells remain poorly elucidated.
Accumulating studies have revealed that the PI3K/AKT/mTOR pathway participates in regulating cellular events, such as cell growth, adhesion, migration and survival [
34‐
37]. Activation of AKT signalling can contribute to cell proliferation and tumor progression by modulating its downstream cell cycle factors [
38]. Furthermore, activated AKT induced the phosphorylation of various downstream targets, such as mTOR, FOXO1 and GSK-3β [
39‐
41]. It has been validated that mTOR inhibitors induced cell cycle arrest and suppressed cell proliferation in EBV associated T- and NK-cell lymphomas [
42]. Recent evidence has supported that inhibition of mTOR contributed to cell cycle arrest in prostate cancer radioresistant cells [
39]. Intriguingly, based on GSEA by TCGA database, we found that HALLMARK_PI3K_AKT_MTOR_SIGNALING was significantly enriched in IMPDH2
high CRC specimens. By qPCR and western blotting, we observed that IMPDH2 could accelerate the G1/S phase transition of CRC cells by regulating expression of cyclin D1, p21Cip1 and p27Kip1. These findings drove us to hypothesize that IMPDH2 might promote cell cycle transition by targeting mTOR to regulate the expression levels of cell cycle regulators. It has been reported that AKT phosphorylation at both Ser473 and Thr308 residues, completely activates the AKT signaling pathway [
43]. LY294002 is a small molecule that competitively and reversibly inhibits the ATP binding site of several different PI3Ks, and is a specific inhibitor of PI3K/AKT pathway. It results in suppression of tumor growth and induction of apoptosis in colon cancer cells, with decreased expression of phosphorylated AKT (Ser473) [
44]. Thus, to further substantiate the above intriguing hypothesis, we examined the levels of p-AKT (Ser473) and p-mTOR. In our study, p-AKT and p-mTOR were found to be downregulated in IMPDH2-silenced CRC cells, but upregulated in IMPDH2-overexpressed CRC cells. Furthermore, increased expression of p-AKT and p-mTOR was significantly suppressed in IMPDH2-overexpressed CRC cells by treatment with AKT inhibitors, along with a significant decrease in cellular growth and colony formation.
Additionally, FOXO transcription factors were supposed to exert its oncogenic effect by regulating the expression of genes involved in diverse cellular processes including apoptosis, cell proliferation and genotoxic/oxidative stresses [
45,
46]. Given that FOXO1 is one of cell cycle transition-related genes [
21,
47,
48], we attempt to validate whether IMPDH2-mediated cell cycle transition is dependent on the PI3K/AKT/FOXO1 pathway. In the same manner, p-AKT and p-FOXO1 were detected to be markedly decreased in IMPDH2-silenced CRC cells, but increased in IMPDH2-overexpressed CRC cells. Furthermore, AKT inhibitors induced a significant decrease of p-AKT and p-FOXO1 in IMPDH2-overexpressed CRC cells, thereby resulting in cell growth arrest and inhibition of colony formation. These above observations suggest that IMPDH2-induced proliferation and tumorigenesis might be due to accelerating cell cycle transition via activation of the PI3K/AKT/mTOR and PI3K/AKT/FOXO1 pathways.
There is compelling evidence that EMT is mediated by regulating PI3K/AKT/mTOR pathway in some human tumors [
49,
50]. Using the mTOR inhibitor rapamycin, we observed that inactivation of the mTOR pathway in IMPDH2-overexpressed CRC cells led to inhibition of cell invasion and migration. More importantly, the epithelial marker E-cadherin was significantly increased in IMPDH2-overexpressed CRC cells after inhibition of the mTOR activity, whereas the mesenchymal markers Vimentin and Snail were decreased. Therefore, our data support that activation of PI3K/AKT/mTOR signalling pathway was required for IMPDH2-induced invasion, migration and EMT of CRC cells.