Abstract
Purpose
Cancer cells are characterized as the uncontrolled proliferation, which demands high levels of nucleotides that are building blocks for DNA synthesis and replication. CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase) is a trifunctional enzyme that initiates the de novo pyrimidine synthesis, which is normally enhanced in cancer cells to preserve the pyrimidine pool for cell division. Glioma, representing most brain cancer, is highly addicted to nucleotides like pyrimidine to sustain the abnormal growth and proliferation of cells. CAD is previously reported to be dysregulated in glioma, but the underlying mechanism remains unclear.
Methods
The expression of CAD and CHIP (carboxyl terminus of Hsc70-interacting protein) protein in normal brain cells and three glioblastoma (GBM) cell lines were measured by immunoblots. Lentiviruses-mediated expression of target proteins or shRNAs were used to specifically overexpress or knock down CAD and CHIP. Cell counting, colony formation, apoptosis and cell cycle assays were used to assess the roles of CAD and CHIP in GBM cell proliferation and survival. Co-immunoprecipitation and ubiquitination assays were used to examine the interaction of CHIP with CAD and the ubiquitination of CAD. The correlation of CAD and CHIP expression with GBM patients’ survival was obtained by analyzing the GlioVis database.
Results
In this study, we showed that the expression of CAD was upregulated in glioma, which was positively correlated with the tumor grade and survival of glioma patients. Knockdown of CAD robustly inhibited the cell proliferation and colony formation of GBM cells, indicating the essential role of CAD in the pathogenesis of GBM. Mechanistically, we firstly identified that CAD was modified by the K29-linked polyubiquitination, which was mediated by the E3 ubiquitin ligase CHIP. By interacting with and ubiquitinating CAD, CHIP enhanced its proteasomal and lysosomal degradation, which accounted for the anti-proliferative role of CHIP in GBM cells. To sustain the expression of CAD, CHIP is significantly downregulated, which is correlated with the poor prognosis and survival of GBM patients. Notably, the low level of CHIP and high level of CAD overall predict the short survival of GBM patients.
Conclusion
Altogether, these results illustrated the essential role of CAD in GBM and revealed a novel therapeutic strategy for CAD-positive and CHIP-negative cancer.
Keywords: CHIP, CAD, Ubiquitination, Glioblastoma, GBM
Introduction
Originating from the glial cells, glioma is the most common cancer developed in the central nervous system. Based on the current World Health Organization (WHO) classification, glioblastoma (GBM) is defined as the grade IV glioma [1]. The aggressive nature of GBM is evident as the median overall survival of anaplastic glioma is 2–5 years, while that of GBM is only 12–15 months [2–4]. Although treatments including surgery, chemotherapy and radiotherapy have exhibited benefits to some extent, the survival rate of GBM patients has not been significantly improved. This is at least partially due to the gap of knowledge about the pathogenesis of GBM. Therefore, identifying critical or novel biomarkers for GBM is urgently required.
To sustain the abnormal proliferation and growth of tumor cells, metabolic pathways are normally rewired in cancer, such as the upregulation of glycolysis, glutaminolysis and nucleotide synthesis [5, 6]. Recently, it is reported that glioma cells are hypersensitive to drugs that target enzymes involved in the de novo pyrimidine synthesis pathway, indicating the essential roles of these enzymes in glioma progression [7]. CAD (Carbamoyl-phosphate synthetase 2, Aspartate transcarbamylase, and Dihydroorotase) is the rate-limiting enzyme in the de novo pyrimidine synthesis [8, 9]. Numerous studies have shown that active CAD is essential for the generation of pyrimidines and disruption of CAD expression or function results in massive cell death [10]. In nearly all types of cancer, hepatocellular carcinoma and glioblastoma for example, the hyper-upregulated CAD generally predicts an overall poor survival and prognosis, which highlights the fundamental role of CAD in tumorigenesis [11].
Several studies about the regulation of CAD activity are described, including transcription, allostery, and oligomerization [12–15]. In contrast, studies about the post-translational modifications of CAD are limited in phosphorylation. Several growth factors and mitogens activated kinases are reported to mediate CAD phosphorylation. For instance, cyclic AMP-dependent protein kinase, MAPK (mitogen-activated protein kinase) and S6K directly phosphorylate CAD at different amino-acid residues, resulting in the allosteric activation of CAD and subsequent pyrimidine synthesis [16–18]. However, how the protein expression of CAD is regulated in physiological and pathological conditions remains largely unknown, which significantly restricts the development of specific pharmacological inhibitors against CAD.
CHIP (carboxyl terminus of Hsc70-interacting protein), also known as STUB1 (STIP1 homology and U-Box containing protein 1), is intrinsically a U-box dependent E3 ubiquitin ligase [19]. CHIP normally targets substrate proteins with K48- or K63-linked polyubiquitination and subsequently directs them for proteasomal degradation [20, 21]. Although CHIP is originally discovered as a co-chaperon protein, it also functions independent of chaperons like HSP70 and HSC70 [22–24]. By polyubiquitinating around a dozen client substrates, CHIP is characterized as a critical proteostatic controller, which is essential for maintaining the homeostasis and pluripotency of cells [25, 26]. Further, CHIP is widely regarded as a tumor suppressor as it ubiquitinates and degrades several oncogenic proteins, such as PKM2, Myc and Src [27–29]. However, the role of CHIP in glioma is still debatable as stated by several controversial studies [27, 30–33].
In this study, we identified CAD as an essential gene that drove the proliferation and colony formation of GBM cells. Mechanistically, we discovered that CHIP negatively regulated CAD by promoting its K29-linked polyubiquitination and proteasomal and lysosomal degradation, which therefore suppressed GBM cell proliferation and colony formation. Further, the high CAD and low CHIP overall predicted the poor prognosis and survival of GBM patients. Altogether, we revealed the roles and functions of the CHIP-CAD regulatory node in GBM, which provided novel therapeutic targets for GBM patients.
Materials and methods
Cell Culture
Human embryonic kidney 293T (HEK293T) cells, HEB cells and GBM cell lines including U251, U87 and T98G were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin. All these cells were cultured at 37 °C with 5% CO2.
For chemical treatments, cycloheximide (CHX) (MCE, HY-12320), bafilomycin A1 (BafA1) (Selleck, S1413) or MG132 (Selleck, S2619) were dissolved in DMSO (Sigma, D5879). Medium containing MG132, BafA1 or CHX was then used to replace the original medium and cells were cultured in the presence of MG132, BafA1 or CHX for indicated time.
Plasmids
shRNA plasmids were generated by inserting the synthesized shRNA oligonucleotides (Sangon Biotech) into the pLKO.1 lentiviral vector. The sequences for shRNA oligonucleotides were listed in the Table 1. Overexpression plasmids were constructed by cloning the coding sequence of CAD or CHIP from HEK293T cDNA with an N-terminal Flag or HA tag into pCMV and pCDH vectors using primers listed in the Table 1, respectively. Point-mutation plasmids included CHIP-K30A (the 30th lysine mutated to alanine) and CHIP-H260Q (the 260th histidine mutated to glutamine) were constructed by cloning the wild-type CHIP plasmid using mutated primers listed in the Table 1, followed by Dpn I treatment. Truncated CHIP plasmids including CHIP ∆U-box and CHIP ∆TPR and truncated CAD plasmids including GLN/CPS (1-1461), DHO/ATC (1457–2225), GLN (1-373), CPSA (391–939), CPSB (929–1461), DHO (1457–1788), and ATC (1911–2225) were constructed by cloning wild-type CHIP plasmid with a Flag tag into pCMV vector and cloning wild-type CAD plasmid with a HA tag into pCMV vector. The sequences of all plasmids were confirmed by direct Sanger sequencing.
Table 1.
Summary of PCR primers, overexpression plasmid cloning primers and shRNAs
| RT-qPCR primers | |
| CAD-F | 5’-TAGTCCTTGGCTCTGGCGTCTA-3’ |
| CAD-R | 5’-TAGTCGGTGCTGACTGTCTCTG-3’ |
| CHIP-F | 5’-TCAAGGAGCAGGGCAATCGTCT-3’ |
| CHIP-R | 5’-GCATCTTCAGGTAGCACAAGGC-3’ |
| β-actin-F | 5’-TTGCCGACAGGATGCAGAAG-3’ |
| β-actin-R | 5’-GTACTTGCGCTCAGGAGGAG-3’ |
| shRNA primers | |
| shCHIP#1 | 5’-GCAGTCTGTGAAGGCGCACTT-3’ |
| shCHIP#2 | 5’-CCCAAGTTCTGCTGTTGGACT-3’ |
| shCAD#1 | 5’-CGAATCCAGAAGGAACGATTT-3’ |
| shCAD#2 | 5’-GCTCCGAAAGATGGGATATAA-3’ |
| shHSP70 | 5’-CACGGCAAGGTGGAGATCATC-3’ |
| sgRNA primers | |
| sgCHIP | 5’-GTTCTGGGCCGAAAGTACC-3’ |
| Primers for cloning genes | |
| CHIP-WT-F | 5’-GCCATGAAGGGCAAGGAG-3’ |
| CHIP-WT-R | 5’-TCAGTAGTCCTCCACCCAGCC-3’ |
| CHIP-H260Q-F | 5’-AAGGACATCGAGGAGCAACTGCAGCGTGTGGGT-3’ |
| CHIP-H260Q-R | 5’-AAGGACATCGAGGAGCAACTGCAGCGTGTGGGT-3’ |
| CHIP-K30A-F | 5’-AGCGCGCAGGAGCTCGCGGAGCAGGGCAAT-3’ |
| CHIP-K30A-R | 5’-ATTGCCCTGCTCCGCGAGCTCCTGCGCGCT-3’ |
| CHIP ∆U-box-R | 5’-GCCATGAAGGGCAAGGAG-3’ |
| CHIP ∆U-box-F | 5’-AATGCAGGCCTGCTGGGC-3’ |
| CHIP ∆TPR-R | 5’-GAGGAGCGGCGCATCCACCAG-3’ |
| CHIP ∆U-TPR-F | 5’-GTAGTCCTCCACCCAGCC-3’ |
| CAD GLN-F | 5’-ATGGCGGCCCTAGTGTTG-3’ |
| CAD GLN-R | 5’-CCGCTCTCTAACTGTCTG-3’ |
| CAD GLN/CPS-F | 5’-ATGGCGGCCCTAGTGTTG-3’ |
| CAD GLN/CPS-R | 5’-AAGCTTTTGGGAGGTCAT-3’ |
| CAD CPS A-F | 5’-CCACCACCACGAAAGGTT-3’ |
| CAD CPS A-R | 5’-GCCAAGGACTAGGACATG-3’ |
| CAD CPS B-F | 5’-ACCTTTCGAACACCTCAT-3’ |
| CAD CPS B-R | 5’-AAGCTTTTGGGAGGTCAT-3’ |
| CAD DHO-F | 5’-ACCTCCCAAAAGCTTGTG-3’ |
| CAD DHO-R | 5’-CAGGACCACACGGCGGAC-3’ |
| CAD ATC-F | 5’-CACCCCCAGACCTCACCC-3’ |
| CAD ATC-R | 5’-GAAACGGCCCAGCACGGT-3’ |
| CAD DHO/ATC-F | 5’-ACCTCCCAAAAGCTTGTG-3’ |
| CAD DHO/ATC-R | 5’-GAAACGGCCCAGCACGGT-3’ |
Lentivirus-mediated gene overexpression and knockdown
Lentivirus production and infection were performed as previously described [34]. Briefly, HEK293T cells were transfected with the lentiviral packaging plasmids (pMD2.G and psPAX2) together with target plasmids by PEI (Polysciences, 23966-2). At 48 and 72 h post transfection, the supernatant was harvested and then centrifuged at 3000 g for 15 min, which was subsequently used to infect cells in the presence of 8 µg/mL polybrene (Sigma, TR-1003). Cells were then subjected to spinning infection by lentiviruses at 1500 rpm at room temperature for 1 h. The overexpression or knockdown efficiency was confirmed by RT-qPCR and immunoblotting analysis at day 2 and day 3 post-infection, respectively. The primer sequences used for the RT-qPCR is listed in the Table 1.
Antibodies
Primary antibodies included CHIP (CST, 2080S), HA (Bimake, A5969), Myc (Bimake, A5968), β-actin (Bimake, A5538), CAD (ABclonal, A8344), Flag (Sigma, F3165), HSP70 (Abclonal, A12948), and HSC70 (Abbkine, ABP51507). The concentrations of primary antibodies used were listed as followed: Flag (1:1000), HA (1:1000), Myc (1:1000), β-actin (1:10000), HSP70 (1:1000), HSC70 (1:2000), CHIP (1:2000), and CAD (1:2000).
Immunoblotting analysis and immunoprecipitation
Cells were lysed in 1xNP40 lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP40, 5 mM EDTA, pH 7.4) that was supplemented with a complete protease inhibitor cocktail (Bimake, B14001), followed by centrifugation at 4 °C for 15 min. Around 10% of the supernatant was prepared as the whole cell lysates (WCL), while the remaining was immunoprecipitated with either anti-Flag (Bimake, B26102) or anti-HA (Bimake, B26202) magnetic beads at 4 °C. The magnetic beads were then washed three times with 1xNP40 buffer and the immunoprecipates were eluted with 1xSDS sample buffer by boiling at 95℃ for 5 min. Eluants were then separated on 6% or 10% SDS-PAGE and electro-transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). After blocking with 3% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4℃ with indicated primary antibodies, followed by incubation with anti-mouse (Affinity, S0002) or anti-rabbit (Affinity, S0001) secondary antibodies at room temperature for 1 h. Membranes were subsequently visualized by Odyssey® infrared imaging system (LI-COR Bioscience).
Quantitative real-time PCR
Total RNA was extracted using the TransZol Up kit (TransGen Biotech, ET111-01) based on the manufacturer’s instructions. Total RNA was subjected to reverse transcription using the cDNA Synthesis kit (TransGen Biotech, AU341-02). qPCR was performed using the SYBR Green kit (TransGen Biotech, AQ601-01). Relative mRNA expression levels of target genes were calculated after normalizing to β-actin using the following formula: 2−ΔΔCt.
Cell proliferation assay
To validate the effects of CHIP and CAD on GBM cell proliferation, 2 × 104 U87 or U251 cells were seeded into a single well of the 24-well plate and then infected with indicated lentiviruses that either knocked down or overexpressed CAD or CHIP. At day 1, 2, 3 and 4 post-seeding, cells were collected by trypsinization and counted using a hemocytometer.
Colony formation assay
1 × 103 U251 and U87 cells with either CAD or CHIP knockdown or overexpression were seeded into a single well of the 6-well plate and then were continually cultured for 2 weeks. When colonies grew to about 1 mm in diameter, the medium was aspirated, and cells were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. The number of colonies were quantitated by Image J software.
Apoptosis assay
Cells were harvested and washed with cold PBS twice and then resuspended in 1xbinding buffer, followed by Annexin V and propidium iodide (PI) staining at room temperature for 15 min (Yeasen Biotechnology, 40302ES). The apoptotic cells were then detected by flow cytometry (Thermo Fisher). Data were further analyzed by FlowJo V10.
Cell cycle analysis
Cells were trypsinized, washed twice with PBS, and then fixed with 70% cold ethanol for 30 min, followed by the treatment of 10 mg/mL RNase (Solarbio, R8021) at room temperature for 10 min. Subsequently, cells were incubated with 10 µg/mL PI in dark for 15 min. Cells were then washed once with PBS to remove the unbound PI and analyzed by flow cytometer (Thermo Fisher). Cell cycle distribution was further analyzed by FlowJo V10.
Statistical analysis
Data were presented as mean ± SEM and analyzed by unpaired two-tailed Student’s t test when two groups were compared and by one-way ANOVA followed by Tukey post hoc test when three or more than three groups were compared. All statistical analyses were performed by GraphPad Prism 5.0. P value < 0.05 is statistically significant. Statistical symbols ‘*’ represents P value < 0.05, ‘**’ represents P value < 0.01, ‘***’ represents P value < 0.001, and ‘n.s.’ indicates ‘not significant’, respectively.
Results
CAD promotes GBM cell proliferation
To determine the role of CAD in glioma, we correlated CAD mRNA expression with prognosis using the GlioVis database. The survival analysis revealed that glioma patients with high CAD exhibited poor prognosis (Fig. 1A). By histology, gliomas are classified as glioblastoma (GBM), astrocytoma (Astro), oligodendroglioma (Oligd) and oligoastrocytoma (Oliga). Significantly, CAD showed high expression in all types of gliomas in comparison to normal adjacent tissues (Fig. 1B). Besides, all high-grade gliomas had significantly upregulated CAD (Fig. 1C; Table 2). These data indicated the essential role of CAD in glioma development. We further examined the relative expression of CAD in different GBM cell lines and all these cells showed feasibly detectable CAD protein (Fig. 1D). We thus knocked down CAD by lentiviruses mediated expression of specific shRNAs in U87 cells that had relatively high endogenous protein level of CAD (Fig. 1D, E). All these two shRNAs significantly decreased CAD protein expression, which consequently reduced the proliferation of U87 cells (Fig. 1E, F). Notably, the transduction of CAD shRNAs almost completely abolished the colony formation of U87 cells (Fig. 1G, H). In accordance with these results, CAD silencing robustly induced the apoptosis of U87 cells (Fig. 1J). Besides, knockdown of CAD significantly induced the cell cycle arrest (Fig. 1I). These data suggested that CAD was indispensable for GBM cell proliferation and colony formation.
Fig. 1.
CAD promotes GBM proliferation. A The high expression of CAD predicted the poor survival of glioma patients. B-C CAD was upregulated in different histological types (B) and all-grade (C) gliomas compared to normal adjacent tissues. Glioblastoma: GBM; Astrocytoma: Astro; Oligodendroglioma: Oligd; Oligoastrocytoma: Oliga. A, B and C panels were obtained by analyzing the GlioVis database (http://gliovis.bioinfo.cnio.es/). D Immunoblotting analysis of CAD protein levels in different glioma cell lines. E Immunoblotting analysis confirmed the knockdown efficiency of CAD by shRNAs. F CAD knockdown inhibited U87 cell proliferation. G-H, CAD knockdown inhibited the colony formation of U87 cells. Representative images were shown in G and the quantification for panel G was graphed in H. I Knockdown of CAD induced U87 cell cycle arrest at G2/M. J CAD knockdown induced the apoptosis of U87 cells. All the data were shown as mean ± SEM from three independent experiments. **, P < 0.01***, P < 0.001
Table 2.
The relationship of CAD or CHIP expression with the clinicopathological features of glioma patients
| Variables | CAD | CHIP | ||||
|---|---|---|---|---|---|---|
| n = 667cases | n = 667cases | |||||
| Low (%) | High (%) | P value | Low (%) | High (%) | P value | |
| All patients | 573 | 94 | 67 | 600 | ||
| Gender | 0.814 | 0.352 | ||||
| Males | 306 | 49 | 34 | 321 | ||
| Females | 217 | 37 | 31 | 223 | ||
| Age (years) | 0.8668 | 0.85 | ||||
| >65 | 71 | 12 | 8 | 75 | ||
| ≤ 65 | 452 | 74 | 57 | 469 | ||
| Histology | 0.226 | 0.031 | ||||
| GBM | 125 | 27 | 17 | 135 | ||
| Astro | 167 | 27 | 27 | 167 | ||
| Oliga | 118 | 12 | 13 | 117 | ||
| Oligd | 163 | 28 | 10 | 181 | ||
| Grade | < 0.001 | 0.002 | ||||
| II | 210 | 16 | 12 | 214 | ||
| III | 199 | 45 | 36 | 208 | ||
| IV | 122 | 27 | 17 | 132 | ||
| Status* | 0.001 | 0.002 | ||||
| 0 | 382 | 46 | 31 | 397 | ||
| 1 | 191 | 48 | 36 | 203 | ||
| 1P/19q codeletion | 0.250 | < 0.001 | ||||
| Codel | 150 | 19 | 2 | 167 | ||
| Non-codel | 417 | 75 | 63 | 429 | ||
| MGMT promoter status | 0.689 | 0.001 | ||||
| Methylated | 414 | 63 | 38 | 439 | ||
| Unmethylated | 136 | 23 | 28 | 131 | ||
| IDH Status | 0.0265 | < 0.001 | ||||
| mutant | 377 | 51 | 29 | 399 | ||
| WT | 189 | 43 | 38 | 194 | ||
GBM, Glioblastoma. Astro, Oligoastrocytoma. Oligd, oligodendroglioma. Oliga, oligoastrocytoma. WT, wildtype. Status*-0, alive. Status*-1, death. Codel, codeletion. Non-codel, non-codeletion
CHIP is a novel interacting protein of CAD
The activity and expression level are the two facets that determine the functionality of proteins. While the phosphorylation of CAD protein is heavily reported, which is crucial for its enzymatic actives, regulators that control CAD protein stability or expression is unknown. By searching the classic protein-protein interaction database (https://thebiogrid.org/), we found the E3 ubiquitin ligase CHIP might bind with CAD. To justify this possibility, we performed co-immunoprecipitation analysis by using CAD as the bait. Analysis of the immunoprecipitants revealed that both exogenous and endogenous CAD physiologically interacted with CHIP (Fig. 2A, B). To further investigate the underlying mechanism by which CHIP bound with CAD, two CHIP mutants were constructed: one incapable of binding chaperones (CHIP-K30A) and another abortive in ubiquitin ligase activity (CHIP-H260Q). Intriguingly, CHIP-H260Q showed the similar affinity to CAD as the wild-type CHIP, whereases CHIP-K30A failed to interact with CAD (Fig. 2C). To further validate that, we examined CAD interaction with two canonical chaperones including HSP70 and HSC70. While CAD could not interact with HSC70, it showed moderate interaction with HSP70 only in the context of CHIP wildtype (WT) or CHIP-H260Q overexpression, indicating that CHIP might mediate CAD interaction with HSP70 (Fig. 2D). Of note, HSP70 knockdown neither disrupted the interaction of CAD with CHIP nor affected CAD protein expression (Fig. 2E, F). These data together suggested that the interaction of CHIP with CAD relied on other chaperons except HSP70 and HSC70.
Fig. 2.
CHIP is a novel interacting protein of CAD. A HA-tagged CHIP (HA-CHIP) was co-immunoprecipitated with Flag-tagged CAD (Flag-CAD) in HEK293T cells. B HA-CHIP interacted with endogenous CAD in HEK293T cells. C CHIP-K30A, but not -H260Q, abolished the interaction of CHIP with CAD. D CAD interacted with HSP70 only in the context of CHIP wildtype (WT) or CHIP-H260Q overexpression HEK293T cells. E HSP70 knockdown had no effects on the interaction of CHIP with CAD. F HSP70 knockdown had no effect on CAD protein level. G The structural domains of CHIP protein. CHIP-FL: full-length CHIP; ∆U-box: CHIP with U-box domain deleted; ∆TPR: CHIP with TPR domain deleted. H The TPR domain of CHIP was essential for its interaction with CAD. I The domain mapping of CAD. J CHIP bound with multiple domains of CAD.
CHIP is composed of several functional domains. Specifically, three tandem tetratricopeptide repeat (TPR) domains of CHIP mediates its interaction with chaperon proteins including HSP70 and HSC70, while the U-box domain possesses the E3 ubiquitin ligase activity [35, 36]. Hence, both TPR and U-box domains are indispensable for CHIP to be functional. To map the specific domain(s) that mediate(s) the CHIP-CAD interaction, a series of plasmids that expressed truncated CHIP or CAD proteins were generated (Fig. 2G, I). Co-immunoprecipitation experiments demonstrated that deletion of TPR (namely ΔTPR) but not U-box (ΔU-box) domains abolished CHIP interaction with CAD (Fig. 2H). Intriguingly, several fragments of CAD, including CPSII, DHO, ATC, GLN, were found to bind with CHIP, albeit to different extent (Fig. 2I, J). Among them, the ATC domain of CAD exhibited the highest affinity to CHIP than others (Fig. 2J). Altogether, these results illustrated that multiple interfaces of CAD mediated the interaction with the TPR domain of CHIP, which was nevertheless independent of HSP70 and HSC70.
CHIP mediated the K29-linked polyubiquitination of CAD
To examine the outcome of CHIP interaction with CAD, we firstly explored whether CAD is regulated by ubiquitination considering the E3 ligase activity of CHIP. To this end, we first examined the ubiquitination status of CAD. HEK293T cells were co-transfected with HA tagged ubiquitin (HA-Ub) and Flag tagged CAD (Flag-CAD), which was subsequently subjected to MG132 treatment. Remarkably, the exogenous CAD was readily polyubiquitinated in the presence of MG132 (Fig. 3A). Of note, the ectopic expression of CHIP enhanced CAD polyubiquitination (Fig. 3B). Consistently, neither the CHIP-K30A mutant that was unable to bind CAD nor the CHIP-H260Q mutant that has impaired ubiquitin ligase activity promoted CAD ubiquitination (Fig. 3C). Further, deletion of either TPR or U-box domains of CHIP failed to promote CAD polyubiquitination, indicating that both the TPR domain and U-box domain were indispensable for CHIP-mediated ubiquitination of CAD (Fig. 3D). Of note, CHIP knockout significantly reduced CAD ubiquitination, which was further restored by the ectopic expression of wild-type CHIP rather than its truncated mutants including ΔTPR and ΔU-box (Fig. 3E, F).
Fig. 3.
CHIP mediated the K29-linked polyubiquitination of CAD. A CAD protein was poly-ubiquitinated. B The ectopic expression of CHIP enhanced CAD polyubiquitination. C CHIP WT rather than CHIP-K30A and -H260Q mutants promoted CAD polyubiquitination. D Neither deletion of U-box (∆U-box) nor TPR (∆TPR) domains promoted CAD ubiquitination. E CHIP knockdown inhibited CAD ubiquitination. F The full length of CHIP (FL) but not CHIP-∆U-box and CHIP-∆TPR mutants rescued CAD ubiquitination in CHIP knockout HEK293T cells. G CAD was modified by K29-linked polyubiquitination. H CHIP overexpression increased the K29-linked polyubiquitination of CAD in HEK293T cells. I CHIP overexpression increased the K29-linked polyubiquitination of CAD in U87
Covalent conjugation of ubiquitin is an indispensable process that mediates the ubiquitination and degradation of target proteins, which is initiated by the covalent conjugation of the carboxyl terminal glycine in ubiquitin to the lysine in client proteins [37]. Seven lysine residues in ubiquitin are responsible for the formation of polyubiquitin linkage, including K6, K11, K27, K29, K33, K48 and K63 [38]. To specify the ubiquitination linkage for CAD, several ubiquitin mutants that remain only one lysine residue with other six lysine mutated to arginine were obtained, namely Ub-K6, -K11, -K27, -K29, -K33, -K48 and -K63. Further, a ubiquitin mutant with all lysine residues mutated to arginine (Ub-K0) was generated, which therefore abolishes the polyubiquitination of substrates [39]. While the ubiquitin WT consistently increased CAD polyubiquitination, Ub-K0 thoroughly prevented that (Fig. 3G). Importantly, CAD was efficiently labelled by Ub-K29 but not other ubiquitin mutants (Fig. 3G). As expected, CHIP significantly enhanced the K29-linked polyubiquitination of CAD, as the mutation of the 29th lysine residue of ubiquitin to arginine (Ub-K29R) almost completely blocked CAD ubiquitination in HEK293T cells (Fig. 3H). Consistent with that, the overexpression of CHIP significantly promoted the K29-linked polyubiquitination of CAD in U87 cells (Fig. 3I). Together, these results indicated that CHIP effectively promoted the K29-linked polyubiquitination of CAD in GBM.
CHIP mediated the proteasomal and lysosomal degradation of CAD in GBM
While ubiquitin has seven lysine responsible for polyubiquitination linkage, only ubiquitin linkage on defined lysine, such as K29 and K48, targets protein for degradation. To explore the outcome of CHIP mediated K29-linked polyubiquitination of CAD, we firstly examined the relative expression of both CHIP and CAD in GMB cells. As expected, CHIP protein expression was negatively correlated with CAD protein level in GBM cells (Fig. 4A). Of note, CHIP had the lowest while CAD had the highest protein expression in normal brain HEB cells than other three GBM cells (Fig. 4A). Accordingly, the overexpression of CHIP reduced CAD protein level in both HEK293T and U251 cells in a dose-dependent manner, indicating that CHIP facilitated the ubiquitination and hence degradation of CAD in GBM (Fig. 4B, C). In contrast, neither CHIP-H260Q nor CHIP-K30A mutants inhibited CAD protein expression in HEK293T and U87 cells (Fig. 4D, E). Further, deletion of either TPR or U-box domains of CHIP failed to reduce CAD protein expression (Fig. 4F). These results indicated that both the E3 ligase activity of CHIP and the physical interaction of CHIP with CAD were required for CHIP-mediated CAD degradation. More importantly, CAD protein level was consistently increased when CHIP was knocked down in three GBM cell lines including U251, U87 and T98G (Fig. 4G-I). As T98G had the highest endogenous expression of CHIP, CHIP knockdown seemed to have the most dramatic effects on CAD protein expression in T98G cells than U251 and U87 cells. Nevertheless, CHIP knockdown barely affected CAD mRNA expression in all three GBM cell lines (Fig. 4J-L). We then tested if CHIP modulated CAD protein stability. HEK293T cells were co-transfected with Flag-CAD and HA-CHIP, followed by the treatment of cycloheximide (CHX) that is the inhibitor of de novo protein synthesis. The ectopic expression of CHIP dramatically enhanced CAD protein degradation in HEK293T cells (Fig. 4M, N). In accordance with that, CHIP knockdown in U87 cells prolonged the half-life of CAD protein (Fig. 4O, P). Both lysosomal inhibitor bafilomycin A1 (BafA1) and proteasome inhibitor MG132 partially rescued CHIP-mediated degradation of CAD in HEK293 as well as U87 cells, suggesting that both lysosomal and proteasomal pathways contributed to CAD degradation in GBM (Fig. 4Q-S). Collectively, these results illustrated that CHIP enhanced CAD ubiquitination, leading to its proteasome- and lysosome-dependent degradation in GBM.
Fig. 4.
CHIP mediated the proteasomal and lysosomal degradation of CAD in GBM. A Western blotting examination of CHIP and CAD protein expression in normal brain HEB cells and GBM cells including T98G, U251 and U87. B-C CHIP overexpression inhibited CAD protein expression in a dose-dependent manner in HEK293T (B) and U251 cells (C). D-E CHIP WT rather than CHIP-K30A and -H260Q mutants decreased CAD protein level in U87 (D) and HEK293T (E) cells. F The full length of CHIP (FL) but not CHIP-∆U-box and CHIP-∆TPR mutants suppressed CAD protein expression in HEK293 cells. G-I, Knockdown of CHIP substantially increased CAD protein expression in U87 (G), T98G (H) and U251 (I) cells. J-L, CHIP knockdown barely affected CAD mRNA levels in U87 (J), T98G (K) and U251 (L) cells. M-N CHIP overexpression shortened the half-life of CAD protein in HEK293T cells. HEK293T cells expressing Flag-CAD were either transfected with vector control or HA-CHIP and subsequently subjected to CHX treatment (50 µg/mL) for indicated time, followed by examination of CAD expression by Western blot. N was the quantification of CAD protein level in M. O-P CHIP knockdown prolonged the half-life of CAD protein in U87 cells. P was the quantification of CAD protein level in O. Q-R Both BafA1 (Q) and MG132 (R) partially rescued CHIP degradation of CAD in HEK293T cells. HEK293T cells expressing Flag-CAD were either transfected with vector control or HA-CHIP and subsequently subjected to BafA1 (100 nM) or MG132 (20 µg/mL) treatment for indicated time. All the data are shown as mean ± SEM from three independent experiments. S Both BafA1 (100 nM) and MG132 (20 µg/mL) partially rescued CHIP degradation of CAD in U87 cells
CHIP inhibited GBM proliferation and colony formation
Given the essential roles of CAD in cancer progression, we then examined the functions of its negative regulator-CHIP in GBM. In accordance with previous observations, a low level of CHIP was intimately correlated with the overall poor survival of glioma (Fig. 5A). Of note, the low expression of CHIP was observed in glioma than its adjacent normal tissues (Fig. 5B, C). Moreover, CHIP expression was inversely correlated with the grades of glioma (Fig. 5B, C; Table 2). To further corroborate the role of CHIP in GBM, CHIP was transduced into U251 and U87 cells, which dramatically impaired GBM cell proliferation in a dose-dependent manner (Fig. 5D, E). As CHIP-K30A and -H260Q were unable to inhibit CAD, the proliferation of U251 and U81 cells were largely unaffected by these mutants (Fig. 5F). Similarly, while CHIP WT robustly suppressed the colony formation of U251 and U81 cells, CHIP-K30A and -H260Q mutants were much less effective (Fig. 5G, H)). Moreover, overexpression of CHIP WT, but not CHIP-K30A and -H260Q significantly induced the apoptosis of U87 cells (Fig. 5I). Thus, these results indicated that CHIP robustly inhibited the proliferation and colony formation of GBM cells, which relied on both enzymatic activity and substrate binding capacity of CHIP.
Fig. 5.
CHIP inhibited GBM proliferation and colony formation. A Low CHIP expression indicated the poor survival of glioma patients. B-C CHIP was downregulated in all-grade (B) and different histological types (C) of gliomas compared to normal adjacent tissues. A, B and C panels were obtained by analyzing GlioVis database (http://gliovis.bioinfo.cnio.es/). D-E CHIP overexpression inhibited the proliferation of U251 (D) and U87 (E) cells in a dose-dependent manner. F CHIP WT rather than CHIP-K30A and -H260Q mutants suppressed the proliferation of U87 cells. G-H CHIP WT, but not CHIP-K30A and -H260Q mutants, inhibited the colony formation of U87 cells. Representative images were shown in H and the quantification for panel G was graphed in H. I CHIP WT, but not CHIP-K30A and -H260Q mutants, increased the apoptosis of U87 cells. All the data are shown as mean ± SEM from three independent experiments
CHIP suppressed the CAD-driven proliferation and colony formation of GBM cells
Our data so far indicated that both CHIP and CAD played fundamental and opposite roles in regulating the proliferation and colony formation of GBM cells. Further analysis showed that the low CHIP and the high CAD expression concomitantly predicted an overall poor survival of glioma patients (Fig. 6A). We thus examined the dependency of CHIP on CAD in regulating GBM cell proliferation. To achieve that, both CHIP and CAD were silenced in U251 cells. CHIP knockdown consistently upregulated CAD protein level in U251 cells, which was entirely blocked by the transduction of CAD shRNAs (Fig. 6B). In agreement with CAD protein expression, CHIP knockdown increased U251 proliferation, which was counteracted by CAD knockdown, indicating that CHIP functioned upstream of CAD (Fig. 6C). To further confirm that, we manipulated the ectopically expressed CHIP to the extent that only partially reduced the overexpressed CAD (Fig. 6D). As expected, while CAD overexpression significantly promoted the colony formation of U87 cells, the ectopic expression of CHIP robustly inhibited that, which was further partially rescued by overexpressing CAD (Fig. 6E, F)). Taken together, these results revealed that CHIP inhibited the proliferation and colony formation of GBM cells by degrading CAD protein.
Fig. 6.
CHIP suppressed the CAD-driven proliferation and colony formation of GBM cells. A The high expression of CAD and low expression of CHIP together predicted the poor survival of glioma patients. B Immunoblotting analysis of CAD and CHIP protein expressions after transduction of either non-targeting control (SC), CHIP shRNAs, CAD shRNAs or both in U251 cells. C The silencing of CAD overrode the enhanced proliferation of U251 cells by CHIP knockdown. D Immunoblotting analysis of Flag-CAD and Flag-CHIP protein expressions after infecting U87 cells with lentiviruses expressing empty vector (Vec), Flag-CAD, Flag-CHIP or both. E-F CHIP overexpression overrode CAD-driven colony formation of U87 cells. Representative images of colonies were shown in E and the quantification for E was graphed in F. All the data are shown as mean ± SEM from three independent experiments
Discussion
Although glioma has been studied for decades, there are currently no effective therapies. As the rate-limiting enzyme in the de novo pyrimidine synthesis pathway, CAD has attracted significant attention in recent years [40]. In order to meet the abnormal proliferation of cancer cells, the biosynthesis of pyrimidine is always robustly enhanced to meet the high demands of DNA and RNA [11, 41]. Numerous studies have revealed that CAD activity is critical for the preservation of pyrimidine pools, which is the prerequisite of cell division and proliferation [42]. In this study, we revealed that CAD is crucial and essential for the proliferation and colony formation of GBM cells. Firstly, the bioinformatic analysis revealed that CAD was highly upregulated in glioma, which was associated with the overall poor survival of glioma patients. Secondly, CAD knockdown dramatically decreased, while the ectopic expression of CAD enhanced the colony formation of GBM cells. Given the critical roles of CAD in the development of GBM and other types of cancer, the regulation of CAD activity or expression is equally important. It is previously reported that several kinases such as MAPK and S6K directly phosphorylate CAD at multiple amino-acid residues, leading to its increased enzymatic activity and subsequently enhanced pyrimidine biosynthesis [43]. However, the regulatory mechanisms about CAD expression, particularly about its protein expression, is barely reported. Here, we firstly reported that CAD is modified by ubiquitination, which was negatively correlated with its protein level. We further identified the E3 ubiquitin ligase CHIP as the physiological interactor of CAD. By interacting with CAD, CHIP mediates the K29-linked polyubiquitination of CAD, leading to its proteasomal and lysosomal degradation in GBM. By functioning upstream of CAD, CHIP significantly inhibits the proliferation and colony formation of GBM cells. Consistently, the bioinformation analysis indicated that CHIP is downregulated in glioma and the low expression of CHIP predicts the overall poor survival and prognosis of glioma patients.
CHIP is initially regarded as a chaperone dependent E3 ubiquitin ligase as the interaction of CHIP with its substrates are mostly mediated by chaperone proteins such as HSP70 and HSC70 [44, 45]. Later, tremendous evidence demonstrated that CHIP also interacts with target proteins independent of chaperons as well. For example, CHIP directly binds with IRE1 and PTEN in the absence of HSP70 and HSC70 [24, 46]. Similarly, we found that CHIP interacts with CAD independent of both HSC70 and HSP70. Paradoxically, CHIP-K30A that is defective in binding chaperons fails to interact with CAD, demonstrating that the interaction of CHIP with CAD remains chaperon dependent. It would be interesting to determine whether other chaperones such as HSP90 and HSPA8 might mediate CHIP interaction with CAD. Moreover, we observed moderate interaction between HSP70 and CAD, which is nevertheless irrelevant to CHIP bound to CAD, indicating that HSP70 might mediate other E3 ubiquitin ligase(s) to bind with CAD. Normally, CHIP mediates K6-, K48- and K63-linked polyubiquitination of target proteins, leading to protein degradation or changes in protein-protein interactions [22, 39, 47]. In sharp contrast, CHIP merely mediates the K29-linked polyubiquitination of CAD. Further in vitro ubiquitination assay is required to determine that CAD is a direct substrate of CHIP. Also, it will be interesting to pinpoint the specific residues of CAD that are ubiquitinated by CHIP.
By catalyzing the degradation of multiple pro-proliferation proteins, CHIP is reported to suppress the progression of diverse cancers, such as colon cancer, pancreatic cancer and gastric cancer [27, 48–50]. However, the role of CHIP in the development of glioma is still debatable. It was previously reported that CHIP promoted the oncogenesis of glioma both in vitro and in vivo with undetermined mechanisms [30]. In sharp comparison with that, several studies demonstrated the tumor suppressive roles of CHIP in glioma. For instance, CHIP inhibits the progression of rat C6 glioma by inducing the polyubiquitination and subsequently lysosomal degradation of c-Myc [27]. Additionally, CHIP knockdown empowers glioma cells resistant to chemotherapeutic drugs by ubiquitinating and degrading SMYD2 [51]. Furthermore, CHIP suppresses the proliferation of GBM cells via mediating EGFR ubiquitination and degradation [31]. Although emerging evidence showed that neurological diseases, such as GHS (Gordon Holmes syndrome), could arise from CHIP mutations, no specific CHIP mutation associated with GBM has been described so far. In contrast, several regulatory mechanisms that reveal CHIP downregulation in GBM are reported. In detail, CSN6 (COP9 Signalosome Subunit 6) that is upregulated in GBM compared to normal brain tissues, is shown to destabilize CHIP by increasing its self-ubiquitination. Additionally, GBM CBX3 (Chromobox protein homolog 3) that is significantly upregulated in GBM directly suppresses CHIP transcription. All these collaborated the tumor suppressive role of CHIP in GBM. Consistently, we found that CHIP inhibits the proliferation and colony formation of GBM cells by promoting the ubiquitination and degradation of CAD. Further investigations to examine the roles of CHIP-CAD regulatory node in GBM progression in vivo is intriguing.
In conclusion, we revealed a novel post-translational modification for CAD and identified CHIP as the E3 ubiquitin ligase for CAD. By promoting the K29-linked polyubiquitination and degradation of oncogenic CAD, CHIP invariably inhibits the abnormal proliferation and colony formation of GBM cells. Altogether, we identified the CHIP-CAD regulatory node as a novel prognostic marker for GBM, which might be targetable for glioma patients.
Acknowledgements
We thank all lab members for their helps and suggestions.
Authors’ contributions
Experimental design, Guanya Li, Tingting Li; data acquisition and analysis, Guanya Li, Kai Xiao, Jianfang Gao, Yinan Li, Shanping He, Tingting Li; writing and revision, Guanya Li, Tingting Li; supervision, Shanping He, Tingting Li; funding source, Shanping He, Tingting Li. All authors reviewed the manuscript.
Funding
This work was supported by grants from the National Natural Science Foundation of China (8210082627), the Natural Science Foundation for Outstanding Young Scholars of Hunan Province (2022JJ20033) and the Key Project of Education Department of Hunan Province (22A0035) to T. Li to T. Li and Hunan Science and Technology Project (2017XK2020), the Key R&D Program of Hunan Province (2019NK2161) to S. He.
Data Availability
All data related to this study are available within the article.
Declarations
Ethical approval
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Guanya Li and Kai Xiao contributed equally to this work.
Contributor Information
Shanping He, Email: hesp@hunnu.edu.cn.
Tingting Li, Email: tingtingli339@hunnu.edu.cn.
References
- 1.P. Syed et al., Autoantibody Profiling of Glioma Serum Samples to identify biomarkers using human proteome arrays. Sci. Rep. 5, 13895 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.P.Y. Wen, S. Kesari, Malignant gliomas in adults. N Engl. J. Med. 359(5), 492–507 (2008) [DOI] [PubMed] [Google Scholar]
- 3.H.S. Phillips et al., Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of Disease progression, and resemble stages in neurogenesis. Cancer Cell. 9(3), 157–173 (2006) [DOI] [PubMed] [Google Scholar]
- 4.R. Stupp et al., Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 10(5), 459–466 (2009) [DOI] [PubMed] [Google Scholar]
- 5.B. Faubert, A. Solmonson, R.J. DeBerardinis, Metabolic reprogramming and cancer progression. Science. 368(6487), 152– (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.I. Martinez-Reyes, N.S. Chandel, Cancer metabolism: looking forward. Nat. Rev. Cancer. 21(10), 669–680 (2021) [DOI] [PubMed] [Google Scholar]
- 7.D.D. Shi et al., De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma. Cancer Cell. 40(9), 939– (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.G. Li et al., Pyrimidine biosynthetic enzyme CAD: its function, regulation, and diagnostic potential. Int. J. Mol. Sci., 22(19), 10253 (2021) [DOI] [PMC free article] [PubMed]
- 9.J. Shin et al., Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle. Nat. Metabolism. 5(2), 277– (2023) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.J.A. Cox et al., Novel role for carbamoyl phosphate synthetase 2 in cranial sensory circuit formation. Int. J. Dev. Neurosci. 33, 41–48 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.D.A. Ridder et al., Key enzymes in Pyrimidine Synthesis, CAD and CPS1, predict prognosis in Hepatocellular Carcinoma. Cancers (Basel), 13(4), 744 (2021) [DOI] [PMC free article] [PubMed]
- 12.R.J. Miltenberger, K.A. Sukow, P.J. Farnham, An e-box-mediated increase in cad transcription at the G1/S-phase boundary is suppressed by inhibitory c-Myc mutants. Mol. Cell. Biol. 15(5), 2527–2535 (1995) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.J. Shin et al., Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle. Nat. Metab. 5(2), 277–293 (2023) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.L. Lee et al., Oligomeric structure of the multifunctional protein CAD that initiates pyrimidine biosynthesis in mammalian cells. Proc. Natl. Acad. Sci. U S A 82(20), 6802–6806 (1985) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.M. Moreno-Morcillo et al., Structural insight into the core of CAD, the multifunctional protein leading De Novo Pyrimidine Biosynthesis. Structure. 25(6), 912– (2017) [DOI] [PubMed] [Google Scholar]
- 16.I. Ben-Sahra et al., Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science 339(6125), 1323-1328 (2013) [DOI] [PMC free article] [PubMed]
- 17.D.H. Kotsis et al., Protein kinase a phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade. Mol. Cell. Biochem. 301(1–2), 69–81 (2007) [DOI] [PubMed] [Google Scholar]
- 18.L.M. Graves et al., Regulation of carbamoyl phosphate synthetase by MAP kinase. Nature. 403(6767), 328–332 (2000) [DOI] [PubMed] [Google Scholar]
- 19.Y. Sha et al., STUB1 regulates TFEB-induced autophagy-lysosome pathway. EMBO J. 36(17), 2544–2552 (2017) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.C.A. Ballinger et al., Identification of CHIP, a Novel Tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol. Cell Biol. 19(6), 4535–4545 (1999) [DOI] [PMC free article] [PubMed]
- 21.C. Cook et al., Loss of HDAC6, a novel CHIP substrate, alleviates abnormal tau accumulation. Hum. Mol. Genet. 21(13), 2936–2945 (2012) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.S.M. Ronnebaum et al., The ubiquitin ligase CHIP prevents SirT6 degradation through noncanonical ubiquitination. Mol. Cell. Biol. 33(22), 4461–4472 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Y. Kopp et al., CHIP as a membrane-shuttling proteostasis sensor. Elife, 6, e29388 (2017) [DOI] [PMC free article] [PubMed]
- 24.S.F. Ahmed et al., The chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) targets PTEN for proteasomal degradation. J. Biol. Chem. 287(19), 15996–16006 (2012) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.M.M. Al Mamun et al., Stub1 maintains proteostasis of master transcription factors in embryonic stem cells. Cell Rep., 39(10), 110919 (2022) [DOI] [PubMed]
- 26.C.F. Liu et al., Proteostasis by STUB1/HSP70 complex controls sensitivity to androgen receptor targeted therapy in advanced Prostate cancer. Nat. Commun., 9(1), 4700 (2018) [DOI] [PMC free article] [PubMed]
- 27.I. Paul et al., The ubiquitin ligase CHIP regulates c-Myc stability and transcriptional activity. Oncogene. 32(10), 1284–1295 (2013) [DOI] [PubMed] [Google Scholar]
- 28.M. Kajiro et al., The ubiquitin ligase CHIP acts as an upstream regulator of oncogenic pathways. Nat. Cell Biol. 11(3), 312–U190 (2009) [DOI] [PubMed] [Google Scholar]
- 29.G. Zhao et al., DDX39B drives Colorectal cancer progression by promoting the stability and nuclear translocation of PKM2. Signal Transduct. Target. Therapy, 7(1), 275 (2022) [DOI] [PMC free article] [PubMed]
- 30.T. Xu et al., Carboxyl terminus of Hsp70-interacting protein (CHIP) contributes to human glioma oncogenesis. Cancer Sci. 102(5), 959–966 (2011) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.J. Hou et al., CSN6 controls the proliferation and Metastasis of glioblastoma by CHIP-mediated degradation of EGFR. Oncogene. 36(8), 1134–1144 (2017) [DOI] [PubMed] [Google Scholar]
- 32.H.R. Ko et al., P42 Ebp1 regulates the proteasomal degradation of the p85 regulatory subunit of PI3K by recruiting a chaperone-E3 ligase complex HSP70/CHIP. Cell. Death Dis. 5(3), e1131 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.L.W. Sun et al., Corosolic acid attenuates the invasiveness of Glioblastoma Cells by promoting CHIP-Mediated AXL degradation and inhibiting GAS6/AXL/JAK Axis. Cells, 10(11), 2919 (2021) [DOI] [PMC free article] [PubMed]
- 34.T. Li, E. Ju, S.J. Gao, Kaposi sarcoma-associated herpesvirus miRNAs suppress CASTOR1-mediated mTORC1 inhibition to promote tumorigenesis. J. Clin. Invest. 129(8), 3310–3323 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.T.Y. Wang et al., The E3 Ubiquitin Ligase CHIP in Normal cell Function and in Disease Conditions, Ann N Y Acad Sci. 1460(1), 3-10 (2020) [DOI] [PMC free article] [PubMed]
- 36.K. Radovanac et al., Stabilization of integrin-linked kinase by the Hsp90-CHIP axis impacts cellular force generation, migration and the fibrotic response. EMBO J. 32(10), 1409–1424 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.K.N. Swatek, D. Komander, Ubiquitin Modifications. Cell Research. 26(4), 399–422 (2016) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.I. Dikic, B.A. Schulman, An expanded lexicon for the ubiquitin code. Nat. Rev. Mol. Cell Biol. 24(4), 273–287 (2023) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.T. Li et al., RNF167 activates mTORC1 and promotes tumorigenesis by targeting CASTOR1 for ubiquitination and degradation. Nat. Commun. 12(1), 1055 (2021) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.G. Herve, Structural insight into the core of CAD. Structure. 25(6), 819–820 (2017) [DOI] [PubMed] [Google Scholar]
- 41.T.P. Mathews et al., Human phospholipase D activity transiently regulates pyrimidine biosynthesis in malignant gliomas. ACS Chem. Biol. 10(5), 1258–1268 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.S. Rabinovich et al., Diversion of aspartate in ASS1-deficient tumours fosters de novo pyrimidine synthesis. Nature. 527(7578), 379–383 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.G.R. Buel, S.G. Kim, J. Blenis, mTORC1 signaling Aids in CADalyzing pyrimidine biosynthesis. Cell. Metab. 17(5), 633–635 (2013) [DOI] [PubMed] [Google Scholar]
- 44.L. Petrucelli et al., CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum. Mol. Genet. 13(7), 703–714 (2004) [DOI] [PubMed] [Google Scholar]
- 45.P. Connell et al., The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat. Cell. Biol. 3(1), 93–96 (2001) [DOI] [PubMed] [Google Scholar]
- 46.X. Zhu et al., Ubiquitination of inositol-requiring enzyme 1 (IRE1) by the E3 ligase CHIP mediates the IRE1/TRAF2/JNK pathway. J. Biol. Chem. 289(44), 30567–30577 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.M. Yang et al., E3 ubiquitin ligase CHIP facilitates toll-like receptor signaling by recruiting and polyubiquitinating src and atypical PKC{zeta}. J. Exp. Med. 208(10), 2099–2112 (2011) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.T. Wang et al., CHIP is a novel Tumor suppressor in Pancreatic cancer through targeting EGFR. Oncotarget. 5(7), 1969–1986 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.S. Wang et al., CHIP functions as a novel suppressor of tumour angiogenesis with prognostic significance in human gastric cancer. Gut. 62(4), 496–508 (2013) [DOI] [PubMed] [Google Scholar]
- 50.J. Xu et al., E3 ubiquitin ligase CHIP attenuates cellular proliferation and invasion abilities in triple-negative Breast cancer cells. Clin. Exp. Med. 20(1), 109–119 (2020) [DOI] [PubMed] [Google Scholar]
- 51.K. Pan et al., STUB1-SMYD2 Axis regulates Drug Resistance in Glioma cells. J. Mol. Neurosci. 72(9), 2030–2044 (2022) [DOI] [PubMed] [Google Scholar]
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Data Availability Statement
All data related to this study are available within the article.