Expression of Derlin-1 and its effect on expression of autophagy marker genes under endoplasmic reticulum stress in lung cancer cells
- Li Xu†1,
- Zan-Hong Wang†2,
- Dong Xu3,
- Gang Lin3,
- Dai-Rong Li1,
- Tao Wan1 and
- Shu-Liang Guo1Email author
© Xu et al.; licensee BioMed Central Ltd. 2014
Received: 21 January 2014
Accepted: 5 June 2014
Published: 11 June 2014
Recent findings indicated that Derlin-1 has an important function in tumour progression. In this study, we aimed to determine whether Derlin-1 has an oncogene function as a cross-talk molecule with autophagy.
Cancer cells were treated with tunicamycin (TM) for 8 and 24 h. The expression of Derlin-1 and autophagy-related genes was determined by western blot. Autophagy was analysed by fluorescence microscopy after staining the cancer cells with monodansylcadaverine. The interaction between Derlin-1 and other proteins was identified using co-immunoprecipitation assay.
Our study demonstrated high Derlin-1 expression levels in most non-small lung cancer cell lines. Derlin-1 expression was enhanced under endoplasmic reticulum (ER) stress. Previous studies revealed that TM triggers the initiation of autophagy by activating Beclin 1, converting LC3I to LC3II and degrading p62. Knockdown of Derlin-1 did not affect Beclin 1 and LC3II expression but disrupted the degradation of p62 under ER stress, which resulted in the blockage of autophagy flux. Furthermore, Derlin-1 and p62 were observed to interact under ER stress.
This study is the first report about the interaction between Derlin-1 and p62. Derlin-1 may function in tumour progression partially by interacting with p62.
Hypoxia and low sugar microenvironment can induce the growth of malignant tumours. Cancer treatment can induce the endoplasmic reticulum (ER) stress response, and lead to misfolded or unfolded protein aggregation in the ER lumen and intracellular Ca2+ balance disorders. Unfolded or misfolded proteins need to be degraded in the ER to maintain homeostasis. Protein degradation has been established as a major effector that governs the levels of individual proteins. Protein degradation currently has two main pathways, namely, the ubiquitin-proteasome-mediated and autophagy-lysosome-mediated degradation pathways. For many years, researchers believed that the ubiquitin–proteasome system (UPS) and autophagy function independently. They also believed that the UPS and autophagy are fulfilled by distinct molecular effectors, separate subcellularly and act on mutually exclusive substrates. However, recent findings strongly suggested the existence of crosstalk and cooperation between these two degradation pathways [1, 2]. Researchers have identified several active molecules between the UPS and autophagy in both normal and abnormal cells [3–5].
Derlin-1 was originally identified as a US11-interacting protein required for cytomegalovirus-mediated turnover of the class I major histocompatibility complex (MHC) heavy chain 1, as well as an ER membrane-binding site for p97/VCP . The p97 ATPase [also called valosin-containing protein (VCP) or Cdc48 in yeast] and its associated co-factors, Ufd1 and Npl4, interact with VCP-interacting membrane protein, Derlin-1 and poly-ubiquitinated protein to facilitate protein retrotranslocation from the ER lumen to the cytoplasm for degradation by 26S proteasome [6–10]. Recent studies reported that Derlin-1 is overexpressed in carcinomas, such as lung cancer, breast cancer and colon cancer [11–13]. Derlin-1 knockdown was also reported to sensitise breast cancer cells to ER stress-induced apoptosis. These findings demonstrated the potential of Derlin-1 as a new oncogene. The definite molecular mechanisms underlying the retrotranslocation of ER proteins into an oncogene need to be clarified. An increasing body of data showed the link between ER function and autophagy. However, little is known about the relationship between Derlin-1 and autophagy genes. In this study, we explored the effect of Derlin-1 on autophagy-related genes under ER stress in lung cancer cells. Our results show that Derlin-1 knockdown could impair both p62/SQSTM1 (p62) degradation and the interaction between these two molecules. Further investigation is needed to provide additional insight into the potential of Derlin-1 as a therapeutic target.
Derlin-1 expression in lung cancer cell lines
Changes in Derlin-1 expression by tunicamycin (TM)
Meanwhile, we detected autophagy gene Beclin 1, LC3 and p62 expression under ER stress in NCI-H446 cells. As shown in Figure 2, we found that the expression of Beclin 1 and LC3II increased, but p62 expression decreased by TM treatment, which indicates that autophagy was triggered under ER stress. Monodansylcadaverine (MDC) is a special dye marker of autophagy that can be absorbed by cells and selectively combined with an autophagy bubble. Changes in the level of autophagy are demonstrated by fluorescence intensity. As shown in Additional file 1: Figure S1, the level of autophagy significantly increased after TM exposure in NCI-H446 cells. However, dots in the autophagy bubble were not obvious in this case. The same as in A549 cells (data not shown).
Changes in autophagy gene expression after Derlin-1 siRNA transfection
Interaction between Derlin-1 and p62
Derlin-1 was initially reported to mediate the elimination of misfolded proteins from the ER and retrotranslocation of proteins into the cytosol . Derlin-1 is a multifunctional protein. Its depletion in Caenorhabditis elegans results in ER stress . Derlin-1 expression is upregulated by inducers of ER stress in yeast  and C. elegans. Recent studies demonstrated the function of Derlin-1 in human cancers. A study using TMA showed that Derlin-1 is upregulated in six types of human carcinomas, and Derlin-1-targeting antibodies suppress colon tumour growth in isogenic mice . Derlin-1 expression is elevated in breast and lung cancers, and correlated with tumour grade and lymph node metastasis [11, 12]. Notably, Derlin-1 can relieve ER stress-induced apoptosis in breast cancer cells. In our study, we proved that the inhibition of autophagy led to aggravated ER stress, and induced downstream apoptosis in HeLa cells (to be published). We also aimed to determine whether Derlin-1, an ER-resident protein, participates in the regulation of autophagy to exert its oncogene function in tumour progression. Our findings reveal that knockdown of Derlin-1 could disrupt the degradation of p62 under ER stress. An interaction between Derlin-1 and p62 was detected using co-immunoprecipitation assay. This study is the first report about the correlation between Derlin-1 and p62. We also investigated the interplay between Derlin-1 and p62, as well as their effect on autophagy and other related signalling pathways.
First, we detected the expression of Derlin-1 in various lung cancer cells, including small lung cancer cells and non-small lung cancer cells. Our results show that the expression of Derlin-1 was higher in most non-small lung cancer cells than that in small lung cancer cells, especially in highly metastatic lung cancer cell line 95-D. This finding was in agreement with that of previous reports, which showed that Derlin-1 overexpression is associated with lymph node metastasis in human lung and breast cancers [11, 12]. Anti-tumour drugs can induce ER stress in most cancer cells, and promote autophagic cell protection in almost all lung cancer cells . Thus, we determined the expression of Derlin-1 in cells treated with lower levels of TM. TM increased Derlin-1 expression within a short period of time (8 h). Previous studies indicated that autophagy is induced under ER stress. We also observed the induction of autophagy by increased expression of Beclin 1, conversion of LC3I to LC3II and degradation of p62. Although the results show that the expression of these genes had been changed after TM treatment, whether Derlin-1 and autophagy are closely related remains inconclusive. We detected the expression of autophagy-related genes in A549 cells transfected with Derlin-1 siRNA to confirm the effect of Derlin-1 on autophagy. The expression levels of Beclin 1 and LC3II were not influenced by Derlin-1 siRNA transfection, but p62 accumulation was detected after Derlin-1 siRNA transfection. The interaction between Derlin-1 and p62 was initially verified using co-immunoprecipitation assay.
During the main pathway of autophagy, initially appearing membrane cisterns called phagophores capture portions of the cytoplasm in double-membrane autophagosomes. These vesicles then deliver cargo for lysosomal degradation [16, 17]. Beclin 1, LC3II and p62 are considered to be the most important molecules during this process, and they are known as autophagy marker genes. p62 is a multifunctional signalling molecule involved in various cellular pathways. SQSTM1 is a well-known autophagic substrate that is widely used as an indicator of autophagic degradation. p62 facilitates selective autophagic removal of ubiquitinated cargo by binding to both ubiquitin and Atg8 covalently bound to the phagophore and autophagosome membranes [3, 18], suggesting that autophagy has an important function in the degradation of ubiquitinated proteins. Studies showed that Derlin-1 is part of a multi-protein complex that mediates the dislocation, ubiquitination and extraction of ERAD substrates from the ER membrane [19, 20]. Flierman reported that MHC-I molecules are rapidly dislocated and ubiquitinated once captured by Derlin-1 . Therefore, the functional overlap of p62 and Derlin-1 in the degradation of ubiquitinated proteins may indicate their potential links. Our study only preliminarily investigated the suspected link between p62 and Derlin-1, but the underlying complex mechanism needs further investigation.
Our findings indicate higher expression levels of Derlin-1 in non-small lung cancer cells than those in small lung cancer cells. The expression of Derlin-1 and autophagy marker genes was altered by ER stress. Knockdown of Derlin-1 may impair the degradation of p62 under ER stress. The interaction between Derlin-1 and p62 was identified in this study. However, further studies are necessary for exploring the functional relationship between Derlin-1 and p62 to widen our knowledge about Derlin-1 as an oncogene.
Material and methods
Cell lines and cell culture
Calu-3, GLC-82 and LTEP-a-2 cell lines were from ATCC (Manassas, VA) and 95-D, A549, NCI-N460, NCI-H209 and NCI-H446 were from Shanghai Cell Bank of Chinese Academy of Science (Shanghai, China). All human lung cancer cell lines were grown in Dulbecco’s minimal essential medium or RPMI 1640 medium containing 10% fetal bovine serum and 50 units/mL penicillin and 50 μg/mL streptomycin sulfate. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
Tunicamycin (TM) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in Me2SO at concentrations of 2 mg/mL for storage at −20°C. To induce ER stress, cells were treated with 2 μg/mL TM for 8 and 24 hours. Cells were plated 24 hours before TM was added to the fresh medium.
Western blot analysis
For cultured cells, cells were washed twice with PBS and lysed with cold RIPA lysis buffer containing protease inhibitors (PMSF [phenylmethylsulphonyl fluoride] 1 mmol/L and leupeptin 0.1 g/L). Cell lysates were collected from culture plates using a rubber policeman, and protein was collected by centrifugation. Protein concentrations were determined by BCA (bicinchoninic acid) protein assay (Pierce, Rockford, IL, USA). Aliquots of 40 μg of proteins were boiled in 2× loading buffer (0.1 M Tris-Cl, pH 6.8, 4% SDS, 0.2% bromophenyl blue, and 20% glycerol) for 10 minutes, loaded into 10% Tris–HCl polyacrylamide gels, and transferred electrophoretically to Immobilon-P membrane (Millipore Corporation, Billerica, MA, USA). Membranes were incubated with primary antibodies and appropriate HRP secondary antibodies. Membranes were additionally probed with an antibody against β-actin to ensure equal loading of protein between samples. Detection was performed with chemiluminescent agents (Pierce). Derlin-1 (N-20, sc-46913), Beclin1 (H-300, sc-11427), GRP78 (H-129, sc-13968) and β-actin (C4, sc-47778) antibodies were from Santa Cruz Biotechnology, p62 (D5E2, 8025S), and LC3A/B (4108 s) antibodies were from Cell Signaling Technology.
siRNA for derlin-1 was purchased from Santa Cruz Biotechnology (h, sc-60519), which contains a mixture of three siRNAs. Subconfluent proliferating cells in 6-well plates were incubated with 50 nM siRNA in 2 mL of medium containing Lipofectamine 2000 (Invitrogen Corporation) according to the manufacturer’s protocol.
Cells were lysed by lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM NaF, 2 mM NaVO4, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail [Roche]) and sonicated. Cell lysates were centrifuged at 13000 rpm for 15 min at 4°C. The supernatant was incubated with antibodies for 2 h, then 50% A + G agarose beads was added for incubation for 1 h at 4°C. The precipitates were washed 3 times with lysis buffer for western blot analysis.
Li Xu and Zan-Hong Wang co-first author.
- Lamark T, Johansen T: Autophagy: links with the proteasome. Curr Opin Cell Biol. 2010, 22 (2): 192-198. 10.1016/j.ceb.2009.11.002.View ArticlePubMedGoogle Scholar
- Korolchuk VI, Menzies FM, Rubinsztein DC: Mechanisms of cross-talk between the ubiquitin-proteasome and autophagy-lysosome systems. FEBS Lett. 2010, 584 (7): 1393-1398. 10.1016/j.febslet.2009.12.047.View ArticlePubMedGoogle Scholar
- Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H, Johansen T: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol. 2005, 171 (4): 603-614. 10.1083/jcb.200507002.View ArticlePubMed CentralPubMedGoogle Scholar
- Pandey UB, Nie Z, Batlevi Y, McCray BA, Ritson GP, Nedelsky NB, Schwartz SL, DiProspero NA, Knight MA, Schuldiner O, Padmanabhan R, Hild M, Berry DL, Garza D, Hubbert CC, Yao TP, Baehrecke EH, Taylor JP: HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS. Nature. 2007, 447 (7146): 859-863.View ArticlePubMedGoogle Scholar
- Brodsky JL, Scott CM: Tipping the delicate balance: defining how proteasome maturation affects the degradation of a substrate for autophagy and endoplasmic reticulum associated degradation (ERAD). Autophagy. 2007, 3 (6): 623-625.View ArticlePubMed CentralPubMedGoogle Scholar
- Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA: A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature. 2004, 429: 841-847. 10.1038/nature02656.View ArticlePubMedGoogle Scholar
- Lilley BN, Ploegh HL: A membrane protein required for dislocation of misfolded proteins from the ER. Nature. 2004, 429: 834-840. 10.1038/nature02592.View ArticlePubMedGoogle Scholar
- Ye Y, Meyer HH, Rapoport TA: The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature. 2001, 414: 652-656. 10.1038/414652a.View ArticlePubMedGoogle Scholar
- Hitchcock AL, Krebber H, Frietze S, Lin A, Latterich M, Silver PA: The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation. Mol Biol Cell. 2001, 12: 3226-3241. 10.1091/mbc.12.10.3226.View ArticlePubMed CentralPubMedGoogle Scholar
- Isakov E, Stanhill A: Stalled proteasomes are directly relieved by P97 recruitment. J Biol Chem. 2011, 286: 30274-30283. 10.1074/jbc.M111.240309.View ArticlePubMed CentralPubMedGoogle Scholar
- Dong QZ, Wang Y, Tang ZP, Fu L, Li QC, Wang ED, Wang EH: Derlin-1 is overexpressed in Non-small cell lung cancer and promotes cancer cell invasion via EGFR-ERK-mediated up-regulation of MMP-2 and MMP-9. Am J Pathol. 2013, 182: 954-964. 10.1016/j.ajpath.2012.11.019.View ArticlePubMedGoogle Scholar
- Wang J, Hua H, Ran Y, Zhang H, Liu W, Yang Z, Jiang Y: Derlin-1 is overexpressed in human breast carcinoma and protects cancer cells from endoplasmic reticulum stress-induced apoptosis. Breast Cancer Res. 2008, 10: R7-10.1186/bcr1849.View ArticlePubMed CentralPubMedGoogle Scholar
- Ran Y, Hu H, Hu D, Zhou Z, Sun Y, Yu L, Sun L, Pan J, Liu J, Liu T, Yang Z: Derlin-1 is overexpressed on the tumor cell surface and enables antibody-mediated tumor targeting therapy. Clin Cancer Res. 2008, 14: 6538-6545. 10.1158/1078-0432.CCR-08-0476.View ArticlePubMedGoogle Scholar
- Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P: Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER ssociated degradation. Cell. 2000, 101: 249-258. 10.1016/S0092-8674(00)80835-1.View ArticlePubMedGoogle Scholar
- Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K, Shiosaka S, Hammarback JA, Urano F, Imaizumi K: Autophagy is activated for cell survival after endoplasmic reticulum stress[J]. Mol Cell Biol. 2006, 26 (24): 9220-9231. 10.1128/MCB.01453-06.View ArticlePubMed CentralPubMedGoogle Scholar
- Juhasz G, Neufeld TP: Autophagy: a forty-year search for a missing membrane source. PLoS Biol. 2006, 4 (2): e36-10.1371/journal.pbio.0040036.View ArticlePubMed CentralPubMedGoogle Scholar
- Mizushima N, Levine B, Cuervo AM, Klionsky DJ: Autophagy fights disease through cellular self-digestion. Nature. 2008, 451 (7182): 1069-1075. 10.1038/nature06639.View ArticlePubMed CentralPubMedGoogle Scholar
- Nezis IP, Simonsen A, Sagona AP, Finley K, Gaumer S, Contamine D, Rusten TE, Stenmark H, Brech A: Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain. J Cell Biol. 2008, 180 (6): 1065-1071. 10.1083/jcb.200711108.View ArticlePubMed CentralPubMedGoogle Scholar
- Lilley BN, Ploegh HL: Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane. Proc Natl Acad Sci U S A. 2005, 102: 14296-14301. 10.1073/pnas.0505014102.View ArticlePubMed CentralPubMedGoogle Scholar
- Ye Y, Shibata Y, Kikkert M, van Voorden S, Wiertz E, Rapoport TA: Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane. Proc Natl Acad Sci U S A. 2005, 102: 14132-14138. 10.1073/pnas.0505006102.View ArticlePubMed CentralPubMedGoogle Scholar
- Flierman D, Coleman CS, Pickart CM, Rapoport TA, Chau V: E2-25 K mediates US11-triggered retro-translocation of MHC class I heavy chains in a permeabilized cell system. Proc Natl Acad Sci U S A. 2006, 103: 11589-11594. 10.1073/pnas.0605215103.View ArticlePubMed CentralPubMedGoogle Scholar
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