Background
Ubiquitination mediated by the ubiquitin–proteasome system is basically required for the protein homeostasis in the cells [
1]. The conjugation of ubiquitin to substrates usually involves three key steps, an activation step initiated by E1, an intermediate step covalently linking ubiquitin to a conjugating enzyme (E2), and a final step usually facilitated by a ligase enzyme (E3) which catalyzes the transfer of ubiquitin from the E2 to the protein substrate [
2]. Members of the E2 family are key components for the ubiquitin–proteasome system. However, the role of the E2 family in autophagy and colorectal cancer (CRC) progression remains poorly defined.
Autophagy is an alternative mechanism to maintain cellular homeostasis, which is characterized by an autophagosome-dependent lysosomal degradation of long-lived proteins and unneeded organelles [
3]. Although autophagy and proteasomal mediated lysosomal degradation use distinct components, they may also have some shared specific mechanisms [
4‐
7]. Recently, the roles of autophagy in malignant transformation and cancer progression are concerned [
8‐
18]. However, the roles of autophagy in tumorigenesis and progression are still not well characterized.
Ubiquitin-conjugating E2 enzyme variant proteins constitute a distinct subfamily within the E2 protein family, which lack the conserved cysteine residue needed for the catalytic activity of E2s [
19,
20]. The functions of ubiquitin-conjugating E2 enzyme variant proteins are not well understood. Ubiquitin-conjugating E2 enzyme variant 1 (Ube2v1), one member of ubiquitin-conjugating E2 enzyme variant proteins, has been controversially suggested as both a candidate oncogene or a tumor suppressor [
19,
21‐
23]. In addition, Ube2v1 has been found as one of the key components of TRAF6 to control NF-κB activation [
20,
24‐
28]. Nevertheless, the role of Ube2v1 in autophagy and cancer including CRC and the mechanisms involved are still largely unknown.
Here, we reported that Ube2v1 promoted ubiquitination and degradation of Sirt1 by the help of Ubc13, inhibited histone H4 lysine 16 acetylation, and finally epigenetically suppressed gene expression of autophagy genes. Importantly, Ube2v1 promoted epithelial mesenchymal transition (EMT) and metastasis using an autophagy-related mechanism. Ube2v1 is employed as a new effective therapeutic target for CRC.
Methods
Cell lines and reagents
The human CRC cell lines DLD-1, SW480, and HCT116 (Additional file
1) were obtained from the Cell Bank at the Chinese Academy of Sciences (Shanghai, People’s Republic of China) and cultured in RPMI-1640 or DMEM containing 10% fetal bovine serum. Human Ube2v1 cDNA were cloned into the GV218 expression vector (Genechem) and GV248 expression vector (Genechem), respectively. Stable overexpression and low expression Ube2v1 cell lines were selected by puromycin (1 mg/ml) and confirmed by RT-qPCR and western blots. Cells were transfected using the Lipofectamine 2000 (Invitrogen, CA, USA). siRNAs (RiboBio silencer siRNA: negative control, Ube2v1 [ID#siG1463152832 and siG1463152844]), Ubc13 (ID#stB000527A and ID#stB0005287B), Sirt1 (ID#siB09917110134 and ID#siB09917110218), ATG5 (ID#siB07103014081), ATG7 (ID#siB101210103531) were transfected at a final concentration of 10 nM using Lipofectamine™RNAiMAX (Invitrogen). 3-MA (SigmaAldrich) was dissolved in DMSO (50 mM stock solution) and always used at a final concentration of 5 mM unless otherwise indicated. BafA1 was diluted in dimethylsulfoxide (DMSO) and used at a final working concentration of 1 μmol/l for 6 and 12 h, respectively. NSC697923 (Selleckchem), specifically inhibiting the activity of Ubc13-Uev1A, was dissolved in DMSO and used at 0.5 and 1 μM for 5 min. The proteasome inhibitor MG132 (Sigma Aldrich) was dissolved in DMSO and added in media at a final concentration of 10 μM for 4 h. Cycloheximide (CHX, Cell Signaling Technology) was added in the culture medium at concentrations of 100 μg/ml for 3, 6, and 9 h, respectively. Sirt1 expression construct has been described previously [
29]. The constructs for GFP-LC3, pCMV-myc-Ube2v1, GFP-Ubc13, pCMV-myc-Ubc13, hemagglutinin (HA)-tagged ubiquitin gene (HA-Ub), and mCherry-GFP-LC3B were generated by PCR and confirmed by sequencing.
To analyze lung metastases, CRC cells (1 × 107/0.2 ml) were injected into the lateral tail vein of 6-week-old BALB/C-nu mice. Ten weeks after tail vein injection, mice were sacrificed, and the lungs were weighed. The lung metastases were examined using H&E staining. The number of surface metastases per lung was determined under a dissecting microscope. For comparison, we also prepared rapamycin for IP injection. Rapamycin (Tokyo Chemical Industry) was dissolved in ethanol, which was then diluted with PBS to a final concentration of 1 mg/ml directly before use. Mice were administered daily via intraperitoneal injections of 5 mg/kg rapamycin for 3 days per week following injection of SW480/Ube2v1 into the lateral tail vein for 2 weeks. Lung pairs were prepared for immunohistochemistry. All animal protocols were performed with the approval of the ethics committee of the Soochow University.
The wound-healing assay
The cancer cells were cultured in 6-well plates and grown in medium containing 10% FBS to nearly confluent cell monolayer, then carefully scratched using a plastic pipette tip to draw a linear “wound” in the cell monolayer of each well. The monolayer was washed twice with PBS to remove debris or the detached cells from the monolayer. The cells were incubated at 37 °C and monitored by time lapse (photographed per 20 min for 12 h) in the Nikon microscope Ti-S (Japan). Under the microscope, the number of cells that migrated into the cell-free zone, base on the zero line of the linear “wound,” was evaluated. The experiments were performed thrice in triplicate and were counted double blind by at least two investigators.
Transwell migration assay
For transwell migration assays, 5 × 105 cells were plated in the top chamber (serum-free medium) onto the non-coated membrane (24-well insert; pore size, 8 μm; Corning Costar) and allowed to migrate toward 10% serum-containing medium in the lower chamber. Cells were fixed after 36 h of incubation with methanol and stained with Giemsa solution. The number of cells invading through the membrane was counted under a light microscope (× 40, three random fields per well).
Transwell invasion assay
For invasion assay, 5 × 105 cells were plated in the top chamber onto the Matrigel-coated membrane (24-well insert; pore size, 8 μm; Corning Costar). Each well was coated freshly with Matrigel (60 μg; BD Bioscience) before the invasion assay. Cells were plated in medium without serum or growth factors, and medium supplemented with serum was used as a chemoattractant in the lower chamber. The cells were incubated for 48 h, and cells that did invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were fixed with methanol and stained with Giemsa solution. The number of cells invading through the membrane was counted under a light microscope (× 40, three random fields per well).
Human CRC samples
Surgically resected CRC specimens with paired normal mucosal counterparts were obtained from The First Affiliated Hospital of Soochow University. All procedures involving human tumor biopsies were performed with the approval of the ethics committee of the Soochow University. The patients had given written informed consent.
RNA isolation and qPCR
Total RNA was isolated using the Trizol (Invitrogen) according to the manufacturer’s instructions. For mRNA, cDNA was generated from 1 μg total RNA per sample using the Transcriptor First Strand cDNA Synthesis Kit (Roche). qPCR was performed by using the ABI StepOne Plus and the SYBR® Select Master Mix (ABI). mRNA expression was normalized using detection of 18S ribosomal RNA. Results are represented as fold induction using the ΔΔCt method with the control set to 1. The sequences of qPCR primers are listed in Additional file
2: Table S1.
Western blot analysis and antibodies
Cell lysates were collected in RIPA lysis buffer (1% Triton-X-100, 20 mM Tris, pH 7.5, 137 mM NaCl, 1 mM EGTA, 10% glycerol, 1.5 mM MgCl2, and protease inhibitor mixture and phosphatase inhibitors; latter 2 were from Roche). Lysates were sonicated and centrifuged at 4 °C. Per lane, whole-cell lysate was separated on 12% SDS-acrylamide gels and transferred on Immobilon PVDF membranes (Millipore). The membranes were probed with primary antibodies overnight at 4 °C and incubated for 1 h with secondary peroxidase-conjugated antibodies (CST). Chemiluminescent signals were then developed with Lumiglo reagent (Cell Signaling Technology) and detected by the ChemiDoc XRS gel documentation system (Bio-rad). Antibodies include anti-Ube2v1 (Abcam, monoclonal ab151725), anti-E-cadherin (Santa Cruz, monoclonal sc-21791), anti-N-cadherin (Boster, polyclonal BA0673), anti-Fibronectin (Boster, polyclonal BA1771), anti-Vimentin (Abcam, monoclonal, ab8978), anti-β-Catenin (Cell Signaling Technology, monoclonal #8480), anti-Twist1 (Proteintech, 25465), anti-Snai1 (Bioss, bs-2441R), anti-LC3B (CST, monoclonal 3868), anti-SQSTM1/p62 (CST, polyclonal 5114), anti-H4K16ac (Immunoway, polyclonal YM3317), anti-Beclin1 (Boster, polyclonal PB0014), anti-histone H3 (Abcam, polyclonal ab1791), and anti-Sirt1 (Santa Cruz, monoclonal sc-74504).
Co-immunoprecipitation and ubiquitination assays
Antibodies include anti-Sirt1 (Santa Cruz, polyclonal sc-15404), anti-HA-probe (Santa Cruz, polyclonal sc-805), anti-Myc (Proteintech Group, Inc., 10828-1-AP), anti-GFP (GeneTex, Inc., Monoclonal GT859), anti-Ubc13 (Cell Signaling Technology, Monoclonal #6999), and anti-IgG (Santa Cruz, polyclonal sc-66931). SW480 cells were lysed in Tris/HCl, pH 7.5, buffered with 1% Triton containing protease inhibitors as described above. Supernatant was incubated with appropriate antibody (2 μg) for at least 90 min at 4 °C followed by incubation overnight with Protein A/G-Sepharose beads (GE Healthcare). After overnight incubation, the agarose beads were washed four times with cold lysis buffer, incubated for 10 min at 108 °C with loading buffer, and subjected to SDS-PAGE and western blot analysis. To detect Sirt1 ubiquitination, 10 mM N-ethylmaleimide was included in the lysis buffer containing a protease inhibitor cocktail (Roche, Hongkong, China).
Immunohistochemistry
Paraffin-embedded slides were incubated with primary antibodies: anti-Ube2v1 (Abcam, polyclonal ab88679), anti-E-cadherin (Dako, monoclonal M3612), anti-Fibronectin (Boster, polyclonal BA1771), anti-Vimentin (Abcam, monoclonal, ab8978), anti-β-catenin (Cell Signaling Technology, monoclonal #8480), anti-SQSTM1/p62 (CST, polyclonal 5114), and anti-Beclin1 (Boster, polyclonal PB0014). Staining was done on a SPlink Detection Kit (SP-9000). We quantified staining intensity and percentage of stained cells. Positive tumor cells were quantified by two independent observers. The staining intensity was scored on a scale of 0–3 as negative (0), weak (1), medium (2), or strong (3). The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tumor area, was scored on a scale of 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). An overall protein expression score (overall score range, 0–12) was calculated by multiplying the intensity and extent positively scores.
Immunofluorescence microscopy
Cells were permeabilized with 0.3% Triton X-100 for 10 min followed by fixation with 2–4% Methanal for 15 min, and blocked with 3% sheep serum at room temperature for 60 min. Then, probed with primary antibodies anti-LC3B, anti-E-cadherin, anti-β-catenin, anti-Vimentine, and anti-SQSTM1/p62 were described before overnight at room temperature, and cells were washed three times with PBS. Stained with anti-rabbit IgG H&L (FITC) (abcam #ab6717) and anti-mouse IgG H&L (FITC) (abcam #ab6785) for 1 h at room temperature, and then the cells were washed three times with PBS. SW480 cells expressing GFP-RFP-LC3 were treated with Torin (Cayman Chemical) 250 nM for 6 h. Nuclei were visualized by staining with DAPI (Sigma Aldrich, USA) for 2 min. The stained cells were observed with an inverted fluorescence microscope (Nikon Ni-U). Autophagy was measured by quantitation of GFP-LC3 puncta per cell using fluorescence microscopy. All GFP-LC3 puncta quantitation was performed by an observer blinded to experimental condition.
Electron microscopy
Cells plated at 2 × 106 cells/mL were treated. In a primary case, each sample was fixed with 2% glutaraldehyde–paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 for 2 h and washed three times for 30 min each in 0.1 M PB. Samples were then postfixed with 1% OsO4 dissolved in 0.1 M PB for 2 h and dehydrated in an ascending gradual series (50–100%) of ethanol and infiltrated with propylene oxide. After sectioning and staining with uranyl acetate and lead citrate, they were observed under an electron microscope (JEM 1200EX; JEOL).
Quantitative mass spectrometry
Proteins were extracted from SW480cell lines with stable overexpressioning Ube2v1 and its control. Total protein concentrations were estimated with the bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific Inc. #23227, Waltham, MA, USA). A quantity of 50 mg of protein from each sample was used following the manufacturer’s protocol (Expedeon, San Diego, CA, USA) with a minor modification by substituting urea with triethylammonium bicarbonate (TEAB) buffer for sample washes to avoid the primary amine group containing chemical that would interfere with TMT labeling. Each sample was digested with sequencing-grade trypsin (Promega, Fitchburg, WI, USA) in 500 mM TEAB buffer overnight in an enzyme to substrate ratio of 1:100 (wt:wt) at room temperature with gentle shaking, followed by a second digestion for 4 h with the same amount of trypsin. The digested peptide from different samples were labeled with tandem mass tags (TMT) reagents (Thermo, Pierce Biotechnology) according to the manufacturer’s instruction (TMT 127, 126 for the samples). Briefly, the TMT label reagents were dissolved by anhydrous acetonitrile and carefully added to each digestion products. The reaction was performed for 1 h at room temperature, and hydroxylamine was used to quench the reaction. The TMT-labeled peptides were desalted using the stage tips. For LC-MS/MS analysis, the MS/MS spectra from each LC-MS/MS run were searched against the selected database using an in-house Mascot or Proteome Discovery searching algorithm. Peptides that have 127/126 scores > 1.2 and 126/127 scores < 0.8 were used for protein identification, and MS/MS spectra for all matched peptides were manually interpreted and confirmed. The QMS experiments were repeated for three times, and similar results were obtained.
Statistical analysis
Data were expressed as mean ± SD. Each experiment was performed in at least three repetitions. Student’s t test (unpaired, two-tailed) was used to compare two groups of independent samples. One-way ANOVA was used for multiple comparisons. For analyses of associations of Ube2v1 expression with clinical parameters of CRC patients, the chi-square test was performed. P values of 0.05 or less were considered statistically significant.
Discussion
Ube2v1 (also named as Uev1A), one of ubiquitin-conjugating E2 enzyme variant proteins, belongs to a distinct subfamily of the E2 protein family [
20,
37]. Based on its structure characteristics, Ube2v1 may have unknown functions different from those classic E2s.
Here, we present a critical role of Ube2v1 in autophagy program. We found Ube2v1 can epigenetically regulate autophagy genes. Ube2v1 can globally suppress gene expression of autophagy-related genes. Mechanistically, Ube2v1 promotes ubiquitination and degradation of Sirt1 through Ubc13, subsequently reduces H4K16ac, and suppresses gene expression of autophagy genes epigenetically. It has been shown that H4K16ac is one of the primary histone target of Sirt1 which is a key epigenetic player involved in autophagy. Our study provided a novel mechanistic understanding about the functional regulation of Sirt1 by Ube2v1-Ubc13-mediated ubiquitination. Noteworthily, our study link the classic ubiquitin-proteasome system especially the E2 member with autophagy machine in CRC, suggesting cooperative interaction between these two systems to control cellular homeostasis in CRC. For a long time, Ube2v1-UBC13, as a key components working with TRAF6, controls NF-κB signaling pathways. Recent reports showed that TAK1 kinase and OTUB1 controls NF-κB activation using similar Ube2v1-UBC13/TRAF6 signaling axis. The understanding of Ube2v1-UBC13 in autophagy discovered a new regulatory pathway for autophagy.
Moreover, we also uncovered a key mechanism by which Ube2v1 promotes metastasis of CRC cells. Accumulating evidences demonstrated that cancer cells usually undergo EMT program to facilitate invasion and metastasis [
36,
38‐
40]. Ube2v1 promotes an autophagy-dependent EMT of CRC cells in vitro and in vivo. Our study functionally links the EMT with autophagy program in CRC. Nevertheless, the role of autophagy in cancer is very complex, depending on cell types and specific process involved [
10,
18,
41‐
44]. In general, tumor cell autophagy has evolved to
deal with intracellular and environmental stress, thus favoring tumor growth and progression [
43]. This implies that autophagy may have differential impact in distinct phases of tumorigenesis including metastasis. For example, the role of autophagy in EMT processes is also diverse. Autophagy induction impairs migration and invasion by reversing EMT in glioblastoma cells [
45]. On the contrary, Beclin1 overexpression promoted EMT process through Wnt/β-catenin pathway under starvation [
46]. Moreover, the pan-inhibitor of Aurora kinases danusertib induces autophagy and suppresses EMT in human breast cancer cells [
47]. Autophagy deficiency stabilizes Twist1 to promote EMT [
48]. DEDD interacts with PI3KC3 to activate autophagy and attenuate EMT in human breast cancer [
49,
50]. In liver-specific autophagy-deficient mice (Alb-Cre; ATG7(fl/fl)), autophagy deficiency in vivo reduces epithelial markers’ expression and increases the levels of mesenchymal markers [
51]. In this study, we found that the tumor cells survived and metastasized to distant organs by a suppressive autophagy program.
Our study presented here has illuminated pro-metastatic function of Ube2v1 in CRC. We present a critical role of Ube2v1 in tumor growth and metastasis of CRC in vivo and in vitro. In CRC patients, Ube2v1 expression is elevated in tumor samples especially in advanced TNM staging and correlated with poorer survival of patients. In vivo studies using orthotopic mouse xenograft models of CRC showed that Ube2v1 promotes tumor growth and metastasis. To our knowledge, the role of Ube2v1 in CRC progression and metastasis has not been established. Only several papers have reported its controversial roles in cancer. Ube2v1, first named as CROC-1, was suggesting as a candidate oncogene by transcriptionally activating FOS proto-oncogene [
19]. Inconsistently, Ube2v1 can also function as a tumor suppressor by protecting cells from DNA damage [
21]. And Ube2v1 expression is increased significantly in the early stage for the acquisition of immortality of tumor cells [
22]. Recently, Ube2v1 was reported to mediate matrix metalloproteinase-1 gene regulation through nuclear factor-кB and promote breast cancer metastasis [
23]. Although concrete evidence for the role of Ube2v1 in cancer remains, some inhibitors targeting Ube2v1 pathway have been developed to treat some type of cancers, such as diffuse large B cell lymphoma cells [
33]. Our study presented here has paved the path forward to develop small-molecule inhibitor targeting Ube2v1 for CRC treatment.