DTL promotes cancer progression by PDCD4 ubiquitin-dependent degradation
- 201 Downloads
Ubiquitin E3 ligase CUL4A plays important oncogenic roles in the development of cancers. DTL, one of the CUL4-DDB1 associated factors (DCAFs), may involve in the process of cancer development. Programmed cell death 4 (PDCD4) is a tumor suppressor gene involved in cell apoptosis, transformation, invasion and tumor progression.
Affinity-purification mass spectrometry was used to identify potential DTL interaction proteins. Co-immunoprecipitation (Co-IP) was performed to verify protein interaction between DTL and PDCD4. mRNA levels in cancer cells and tissues were detected by Quantitative real-time PCR. Lentivirus was used to establish stable overexpression and knocking down cell lines for DTL and PDCD4. Transwell and wound healing assays were used to determine migration ability of cancer cells. Matrigel assay was used to determine invasion ability of cancer cells. MTT and colony formation assays were used to evaluate proliferation of cancer cells.
In this study, programmed cell death 4 (PDCD4) was identified as a potential substrate of DTL. Co-IP and immunofluorescence assays further confirmed the interaction between DTL and PDCD4. Moreover, DTL overexpression decreased the protein level and accelerated the degradation rate of PDCD4. Through in vitro ubiquitination experiment, we proved that PDCD4 was degraded by DTL through ubiquitination. Clinically DTL was significantly up-regulated in cancer tissues than that in normal tissues. The survival curves showed that cancer patients with higher DTL expression owned lower survival rate. Functional experiments showed that DTL not only enhanced the proliferation and migration abilities of cancer cells, but also promoted the tumorigenesis in nude mice. Rescued experiment results demonstrated that silencing PDCD4 simultaneous with DTL recovered the phenotypes defect caused by DTL knocking down.
Our results elucidated that DTL enhanced the motility and proliferation of cancer cells through degrading PDCD4 to promote the development of cancers.
KeywordsDTL CUL4A PDCD4 Cancer development
DDB1 cullin4 associated factor
Denticleless E3 ubiquitin protein ligase homolog
Eukaryotic initiation factor-4A
Eukaryotic initiation factor-4G
c-Jun N-terminal kinase
3-(4,5- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide
Non small cell lung cancer
Programmed cell death protein 4
The ubiquitin-proteasome proteolysis system responsible for degrading proteins is involved in nearly all cellular processes . Dysregulation in the components of the ubiquitin system leads to a variety of diseases such as cancers [2, 3]. Cullin protein family containing conserved cullin homology domain is a major type of E3 ligase [4, 5]. CUL4A (Cullin 4A), a member of the cullin protein family, forms CRL4A (cullin 4A–RING ubiquitin ligase) complex with a ring finger protein ROC1, an adapter protein DDB1, and DDB1 cullin4 associated factors (DCAFs) .
In CRL4A complex, DCAFs act as substrate recognizers to mediate the substrate degradation directly. DTL, also called CDT2, DCAF2 or RAMP, is one of the DCAFs containing seven WD40 domains in its N-terminal. DTL is first identified as retinoic acid-regulated nuclear matrix-associated protein (RAMP) since it is down-regulated during RA-induced differentiation of NT2 cells . The roles of DTL in regulating DNA replication and cell cycle were well-illustrated so far . For example, studies in yeast revealed that DTL deletion severely slowed down S-phase progression . DTL substrates, such as Cdt1, PR-Set7/Set8 and p21, render DTL preventing cells from DNA damage in S phase and after UV irradiation [10, 11, 12, 13, 14, 15].
The essential roles of DTL in genomic stability suggest that this ubiquitin system component may involve in tumorigenesis . Remarkably, previous studies showed that DTL expression was elevated in many cancers [17, 18, 19, 20]. Previously studies in breast cancer and Ewing sarcoma showed that DTL knockdown weakened the proliferation and migration abilities of cancer cells [17, 19]. However, the systematic studies on DTL functions in cancers remains to be evaluated. More importantly, the underlying mechanisms of DTL regulating cancer progression need more investigation.
Programmed cell death 4 (PDCD4) is a tumor suppressor gene involved in cell apoptosis, transformation, invasion, and tumor progression [21, 22]. PDCD4 exerts its activities by interacting with eukaryotic translation initiation factors 4A (eIF4A) and 4G (eIF4G) [23, 24]. It has been found that PDCD4 expression deficiency was closely associated with the progression and prognosis of various cancers, such as the myloid leukemia, lung, ovarian, colon and glima cancers [25, 26, 27, 28, 29]. Considering the importance roles of PDCD4 in cancer progression, the regulation of PDCD4 level in cancers deserves more investigation.
In our study, a novel PDCD4 degradation way via ubiquitin system was elucidated. The results of this study suggested that DTL bound with and down-regulated PDCD4 by ubiquitin degradation and promoted the migration, invasion and proliferation abilities of cancer cells. Above all, our data provided novel mechanistic insights into the function of DTL in cancer progression.
Material and methods
Cell lines and cell culture
MCF7, MDA-MB-468 and 293 T cells were preserved by our laboratory and cultured in DMEM with 10% FBS. MDA-MB-231, BT549, SKBR-3, H1650 and A549 cells were preserved by our laboratory and cultured in RPMI-1640 medium with 10% FBS. Human breast epithelial cell line MCF-10A was cultured in DMEM/F12 with 5% horse serum (Biological Industries), 10 μg/ml insulin (Solarbio), 20 ng/ml epidermal growth factor (Pepro Tech), 100 ng/ml cholera toxin (Macgene), 0.5 μg/ml hydrocortisone (Macgene). All cells were grown at 37 °C with 5% CO2/95% air atmosphere and were revived every 3 to 4 months.
Wide type DTL and truncated DTL cDNA sequences were constructed into PLVX-AcGFP-N1 vector. shRNA sequences for DTL and PDCD4 were constructed into pLKO.1-puro vector. shRNA sequences used to establish a conditional MDA-MB-231 DTL silent cell line were constructed into Tet-pLKO-puro plasmid. All the plasmids were transformed and preserved in E.coli strain Stbl3. Human DTL and PDCD4 cDNAs were sub-cloned using I5 polymerase (Tsingke, Qingdao). Primer and shRNA sequences were listed in Additional file 9: Table S1.
Mass spectrometry and protein identification
293 T cells with stable Flag-DTL expression were collected using a scraper and lysed in weak lysis buffer. The cell lysate was incubated with Flag magnetic affinity resin bought from Sigma-Aldrich. SDS-PAGE was performed and gel was stained using Coomassie brilliant blue R250. Protein bands were excised from the gel and then analyzed on LC-MS. Data analysis and protein identification were done by searching against the NCBI protein database.
Establishment of stable cell lines
Constructed overexpression and silencing plasmids were transfected into 293 T cells, together with package plasmids psPAX and PMD2.G at the ration of 4:3:1 using Transfection Reagent Lipofectamine 2000 (Invitrogen). The supernatants were collected and filtered through 0.4 μm filter membrane after 72 h. Cells were plated in 6-well plates with 1 × 106 cells per well and transfected by collected lentivirus particles. The transfected cells were selected by puromycin for 2 weeks. Establishment of stable cell lines was verified by Western blot.
Antibodies and reagents
The antibodies used in our experiments are listed in Additional file 10: Table S2. CHX, MG132 were purchased from Calbiochem, protein G/A from Millipore, protease inhibitor cocktail from Roche, TRIzol and Lipofectamine 2000 from Invitrogen and protein lysis buffer RIPA from Beyotime Biotechnology. Whole cell lysates for immunoblots and immunoprecipitation were prepared using RIPA lysis buffer supplemented with protease inhibitors. mRNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The reverse-transcription reaction was performed using Revert Aid First Strand cDNA Synthesis Kit (Thermo). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green PCR Master Mix (CWBIO) and ABI PRISM 7900HT Real-time PCR Detection System (Eppendorf). Primers used for qRT-PCR were listed in Additional file 11: Table S3.
DTL three-dimension structure was predicted by I-TASSER using ab initio protein structure prediction technique [30, 31, 32]. PDCD4 three-dimension structure was downloaded from Protein Data Bank (http://www.rcsb.org/) using PDB ID 3EIJ . Protein-protein interaction prediction was performed by ZDOCK server . We used GEO datasets GSE3744, GSE10780, GSE19804 and GSE19188 to confirm DTL expression in breast and lung cancers. Of the two affymetrix ID identified, 218585_s_at was selected as the best probe for DTL. Survival curves were drawn by Kaplan Meier plotter, which was a combination of gene expression and patient survival information from GEO (Affymetrix microarrays only), EGA and TCGA database .
Immunohistochemistry and scoring
Samples of breast cancer, NSNLC tissues and corresponding adjacent non-cancerous tissues were obtained from patients undergoing surgical excision of tumors in Qilu Hospital of Shandong University (Jinan, China). The samples used for paraffin sections were fixed with 10% formalin, and the samples for protein and mRNA extractions were frozen in liquid nitrogen immediately after dissection. Research protocols were approved by the Hospital Ethics Committee of Shandong University and written informed consent was obtained from patients based on the Declaration of Helsinki.
Cell proliferation, migration and invasion assays
MTT and colony formation assays were performed to assess cell proliferation as described . Wound-healing, transwell and matrigel assays were used to examine cell motility as described . To avoid the affection of cell proliferation on cell motility, cells for transwell and matrigel assays were cultured in a low-serum concentration (0.2%), cells for wound healing assay were cultured without serum.
Nude mice xenograft and transplanted models
Nude mice (6 weeks old) were purchased from Beijing Huafukang Bioscience Co. INC and maintained in micro-isolator cages in SPF laboratory animal room. All animals were used in accordance with institute guidelines and the experiments were approved by the Use Committee for Animal Care of the institute. For subcutaneous inoculation, cells re-suspended in PBS at a concentration of 2 × 107 cells/mL were injected into 8-week old nude mice. The tumors were measured every 3 days after appearance and the tumor volumes were calculated by the formula (length×width)2/2. For metastatic assay, cells with overexpression or knockdown of DTL and corresponding controls were re-suspended in PBS at a concentration of 1 × 107 cells/mL. Cell suspension (0.1 mL) was injected into tail veins of nude mice. The nude mice were sacrificed by anesthesia with chloral hydrate.
Results are expressed as mean ± SD from at least three independent experiments. SPSS17.0 statistical software package (SPSS Inc.) was used for statistical analysis. Statistical differences between groups were assessed using the Student t test. Statistics of IHC results were calculated using IOD measurement by Image Pro Plus. Association between DTL and PDCD4 expression in breast and lung cancer tissues was evaluated by the Spearman rank correlation test. P < 0.05 was considered statistically significant.
DTL combines directly with PDCD4
DTL degrades PDCD4 through ubiquitin-proteasomal pathway
As phosphorylation of PDCD4 at Ser67 has been proved to play important roles in SCF ligase mediated degradation, we then detected whether phosphorylation of PDCD4 at Ser67 influenced the binding between PDCD4 and DTL. A mutant form of PDCD4 was constructed by substituting Ser67 to Ala67. Alanine was structurally similar to serine, which could reduce phosphorylation level without affecting the structure of PDCD4. As shown in Additional file 3: Figure S3, the interaction between PDCD4(S67A) and DTL was not detected, indicated that the phosphorylation of Ser67 played an important role in its binding with DTL.
To further explore potential functions of DTL in cancers, gene expression profiling on 293 T-DTL and its control cells was employed. Among the 5600 different expression genes identified, several pathways were enriched in GSEA pathway analysis, including cancer related JNK pathway (Fig. 4d). According to the previous study that silencing PDCD4 induced MAP4K1 expression thus gave rise to JNK activation , the impact of DTL on JNK pathway was further explored. As shown in Fig. 4e, DTL overexpression up-regulated the phosphorylation levels of c-JUN and JNK (marked as p-c-JUN and p-JNK). Our results demonstrated that DTL ubiquitination degraded PDCD4 and activated JNK pathway.
DTL is commonly up-regulated in cancer tissues and related to poor outcomes
DTL enhances proliferation, migration and invasion of cancer cells in vitro and in vivo
Moreover, to detect the functions of DTL for cancer cells in vivo, H1650 and MDA-MB-231 cells with DTL overexpression and knocking down were hypodermically injected into nude mice respectively. The results indicated that DTL overexpression promoted tumor growth (Fig. 6h-j) and DTL knockdown inhibited tumor growth in terms of tumor weight and volumes (Fig. 6k-m). To detect the cancer cell mobility in vivo, established breast cancer cell lines with overexpression and silencing DTL were injected into nude mice through the tail vein respectively. Paraffin sections were stained with Hematoxylin-Eosin to identify the metastatic foci. Overexpression DTL significantly increased the number of mice with distant metastasis, while silencing DTL reduced the distant metastasis (Fig. 6n and o). Immunohistochemical analysis showed the expression of PDCD4 levels in mice xenograft tumors were negatively correlated with DTL expression (Additional file 7: Figure S7H).
PDCD4 mediates DTL functions in cancer cells
As described above, we proved that DTL overexpression induced the down-regulation of PDCD4 level and silencing PDCD4 is reported to induce the activation of JNK pathway , we then used JNK inhibitor JNK-IN-8 to detect whether JNK pathway participated in the regulation of cell motility and proliferation abilities in DTL overexpression cells. 5 μM JNK-IN-8 was added into DTL overexpression cells for 3 h (Additional file 8: Figure S8A). MTT and colony formation assays showed that JNK inhibitor reduced the proliferation ability of MCF7 and BT549 cells (Additional file 8: Figure S8B-D). Also, as shown in Additional file 8: Figure S8E and F, cells with JNK inhibitor exhibited lower capability of migration and invasion.
DTL is identified as one of the DCAFs of CUL4A and plays important roles in cell cycle and DNA repair. In our study, DTL overexpression increased PDCD4 ubiquitination level and decreased PDCD4 protein level. Overexpression of DTL accelerated tumor cell growth and increased invasive ability of cancer cells. Moreover, silencing PDCD4 rescued cancer cell growth and mobility deficiencies caused by DTL knockdown. To our knowledge, this is the first report to show that DTL played roles in degrading tumor suppressor PDCD4.
Previous studies showed that CUL4A played a functional role in metastasis in cancer cells by inducing proliferation, EMT, migration, and invasion . DCAFs including DTL interact with DDB1 thus to CUL4A to help receive substrates and exert variable functions . Several substrates of DTL have been identified such as CDT1, P21, PR-Set7/Set8. As essential for proper genome replication [40, 41], CDT1 is considered to play an important role in mediating DTL induced cancer cell proliferation. It is reported DTL depletion in cancer cells caused apoptotic death of cancer cells associated with rereplication due to the loss of CDT1 degradation, but not in non-transformed cells . Here we reported that PDCD4 might be another mediator in DTL regulating cancer cell proliferation.
Our studies revealed that DTL interacted with PDCD4 directly. Sequence analysis showed that DTL has seven WD40 domains in the N-terminal with two WDXR modules. Detailed analysis of protein interaction revealed that PDCD4 bind to WD40 domains of DTL directly. In addition, using ab initio protein structure prediction, we found that DTL has two β-propeller, one of them was formed by WD40 domain in the N-terminal, which has been proved to play important roles in binding with PDCD4. Further studies would be needed for the structural analysis of DTL and DDB1-CUL4A complex.
Programmed cell death 4 (PDCD4) was generally known as tumor repressor inhibiting translation initiation by displacing eIF4G and RNA from eIF4A . PDCD4 was down-regulated or loss in many cancers and related to tumor progression and poor outcomes, such as colon cancer, ovarian cancer, glioblastoma and lung cancer [25, 26, 27, 28]. Previous studies showed that PDCD4 was degraded by two ways, miR-21 and SCFβTRCP mediated degradation [23, 44, 45]. Our study provided evidences that overexpression DTL decreased PDCD4 expression level and promote the ubiquitin level of PDCD4 in cells. In addition, previous studies demonstrated that silencing PDCD4 up-regulates MAP4K1 expression thus to active AP-1 dependent transcription [38, 46]. In this study, we also found that DTL induced MAP4K1 expression and enhanced phosphorylation levels of JNK and c-Jun proteins. These findings suggested that DTL degraded PDCD4 to active JNK pathway.
In conclusion, our study firstly reported the interaction between DTL and PDCD4. DTL overexpression elevated PDCD4 ubiquitin level and accelerated PDCD4 degradation. Functional assays revealed that DTL increased proliferation, migration and invasive abilities of cancer cells, as well as in vivo tumor genesis abilities. Also, PDCD4 recovered cell growth deficiencies caused by DTL knocking down in proliferation and motilities. Our results suggested DTL promote cancer progression through degrading PDCD4.
The authors will thank Qilu hospital for great help in providing tumor tissues.
HC, QW and GW conceived of and designed the study. HC, QW and YS performed the experiments. HC and ZL analyzed and interpreted the data. MF contributed the materials. HC and YW wrote the manuscript. ZZ, YW and GW reviewed and edited the manuscript. All authors read and approved the final manuscript.
This work was supported by the National Key R&D Program of China (No.2017YFC1308600) and National Natural Science Foundation of China (No. 81872148) to Guangwei Wei, by National Natural Science Foundation of China (No. 81874040) and Taishan Scholar Program of Shandong Province to Yunshan Wang.
Ethics approval and consent to participate
Study protocols were approved by the Hospital Ethics Committee of Shandong University and written informed consent was obtained from patients based on the Declaration of Helsinki.
Consent for publication
The authors declare that they have no competing interests.
- 17.Ueki T, Nishidate T, Park JH, Lin ML, Shimo A, Hirata K, et al. Involvement of elevated expression of multiple cell-cycle regulator, DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein), in the growth of breast cancer cells. Oncogene. 2008;27(43):5672–83.PubMedCrossRefGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.