UBL4A inhibits autophagy-mediated proliferation and metastasis of pancreatic ductal adenocarcinoma via targeting LAMP1
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Ubiquitin-like protein 4A (UBL4A) plays a significant role in protein metabolism and the maintenance of cellular homeostasis. In cancer, UBL4A represses tumorigenesis and is involved in various signaling pathways. Pancreatic ductal adenocarcinoma (PDAC) is still a major cause of cancer-related death and the underlying molecular mechanism of UBL4A and PDAC remains unknown.
First, the prognostic role of UBL4A and its expression in human PDAC patients and in pancreatic cancer cell lines were detected by survival analysis and qRT-PCR, western blotting, and immunohistochemistry. Next, the effects of UBL4A on proliferation and metastasis in pancreatic cancer were evaluated by functional assays in vitro and in vivo. In addition, chloroquine was introduced to determine the role of autophagy in UBL4A-related tumor proliferation and metastasis. Ultimately, coimmunoprecipitation was used to confirm the interaction between UBL4A and lysosome associated membrane protein-1 (LAMP1), and western blotting was performed to explore the UBL4A mechanism.
We found that UBL4A was decreased in PDAC and that high levels of UBL4A correlated with a favorable prognosis. We observed that UBL4A inhibited tumor proliferation and metastasis through suppression of autophagy, a critical intracellular catabolic process that reportedly protects cells from nutrient starvation and other stress conditions. UBL4A caused impaired autophagic degradation in vitro, a crucial process in autophagy, by disturbing the function of lysosomes and contributing to autophagosome accumulation. We found a positive correlation between UBL4A and LAMP1. Furthermore, UBL4A caused lysosomal dysfunction by directly interacting with LAMP1, and LAMP1 overexpression reversed the antitumor effects of UBL4A in pancreatic cancer. In addition, we demonstrated that UBL4A suppressed tumor growth and metastasis in a pancreatic orthotopic tumor model.
These findings suggest that UBL4A exerts an antitumor effect on autophagy-related proliferation and metastasis in PDAC by directly targeting LAMP1. Herein, we describe a novel mechanism of UBL4A that suppresses the progression of pancreatic cancer. UBL4A might be a promising target for the treatment and prognostication of PDAC.
KeywordsUbiquitin-like protein 4A, UBL4A Lysosome associated membrane protein-1, LAMP1 Autophagy Lysosome dysfunction Pancreatic ductal adenocarcinoma, PDAC Proliferation and metastasis
Green fluorescent protein
Lysosome associated membrane protein-1
Lysosome associated membrane protein-2
Microtubule-associated protein 1 light chain 3 beta
Pancreatic ductal adenocarcinoma
Real-time quantitative reverse transcription-polymerase chain reaction
Red fluorescent protein
Ubiquitin-like protein 4A
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignant tumors. The 5-year overall survival rate of PDAC is only 9%, despite many large-scale efforts and continuous attempts to improve diagnosis and treatment . The high mortality rate of pancreatic cancer patients is due to the difficulty in diagnosing the disease at an early stage, its tendency to metastasize and its drug resistance [2, 3]. Therefore, early diagnosis of PDAC and identification of appropriate therapeutic targets are of great importance.
Ubiquitin-like protein 4A (UBL4A) is a small protein composed of 157 amino acids located on the X chromosome (Xq28) . Recent studies have indicated that UBL4A acts as a chaperone in protein processing in the endoplasmic reticulum [5, 6]. Other known functions of UBL4A include involvement in tumor suppression and in cell death in response to DNA damage , indicating the versatile capabilities of this protein. However, understanding of the exact biological function of UBL4A in the regulation of cancer cells, especially in PDAC, remains elusive .
Autophagy is a complex catabolic process that engulfs damaged proteins and organelles in autophagosomes and degrades them by fusion with lysosomes to protect cells from nutrient starvation and other stress conditions [9, 10]. Although autophagy was originally identified as a protective mechanism during starvation, it has also been associated with cell death . Thus, autophagy is thought to underlie various processes in oncological diseases by modulating the initiation and/or maintenance of cancers [12, 13]. Lysosomes are responsible for the degradation of macromolecules derived from the extracellular space through endocytosis or phagocytosis, as well as from the cytoplasm through autophagy [14, 15]. Although autophagy has been well described as being directed by proteins encoded by autophagy-related genes (ATGs), there is increasing evidence that lysosomes are the central regulators of the autophagic process . In addition, lysosomal membrane proteins may be involved in the interaction and fusion of lysosomes with autophagosomes, as well as in regulating the stability and integrity of the lysosome [17, 18].
Lysosome associated membrane protein-1 (LAMP1) and LAMP2 are major protein components of the lysosomal membrane. Both of these proteins were originally thought to protect the lysosomal membrane against the action of hydrolytic enzymes . LAMP proteins are important regulators of the successful maturation of both autophagosomes and phagosomes. LAMP2 deficiency causes an accumulation of autophagosomes in many tissues, leading to cardiomyopathy and myopathy in mice and in patients suffering from Danon disease . However, the role of LAMP1 in autophagy remains poorly understood.
In this study, we demonstrated that the elevated expression of UBL4A contributed to a favorable prognosis for PDAC patients and that UBL4A suppressed tumor growth and metastasis by inhibiting autophagy. In addition, we observed that UBL4A caused impaired autophagic degradation by disturbing the function of lysosomes through a direct interaction with LAMP1. Herein, we propose a novel mechanism by which UBL4A suppresses the autophagy-related proliferation and metastasis of PDAC.
Patients and specimens
Clinical correlation between UBL4A mRNA expression and clinical and pathological characteristics in PDAC patients
UBL4A mRNA expression
I + IIa
IIb + III
Flag-tagged lentiviral vectors encoding human UBL4A (Lv-UBL4A-Flag) and empty vectors were constructed in GV341 (GeneChem, Shanghai China). The lentiviruses with scrambled shRNA against UBL4A (Lv-shUBL4A) and the shRNA control (shCtrl) were constructed in GV112 (GeneChem, Shanghai China). After lentiviral infection, single-cell clones were selected by 2.5 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 2 to 4 weeks. The SiRNA for LAMP1 (si-LAMP1) and the siRNA negative control (si-NC) were purchased from RIBOBIO (Guangzhou, China). To induce overexpression of LAMP1, human LAMP1 (NM_005561) cDNA was cloned into a plasmid (GeneCopoeia, Guangzhou, China). For transient transfection, the cells were seeded in six well plates, and 50 nm siRNA or 2 μg plasmids were transfected into the cells using Lipofectamine 2000 (Life Technologies Limited Paisley, Grand Island, NY, USA). The efficiency of all transfections was evaluated by qRT-PCR and/or western blotting. The target sequences of the lentiviruses and siRNAs are listed in Additional file 1: Table S1.
RNA extraction and quantitative real-time PCR analyses
Total RNA was extracted and isolated from the cell lines and frozen tumor specimens using a AxyPrep Multisource Total RNA Miniprep Kit from Axygen (Corning, Suzhou, Jiangsu, China), and the first strand cDNA was synthesized using the Rever TraAce qPCR RT Kit Master Mix with gDNA Remover (FSQ-301, Toyobo Co. Ltd. Osaka, Osaka Prefecture, Japan) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed as previously described . Briefly, qRT-PCR (SYBR Green Assay, Roche Diagnostics GmbH, Indianapolis, IN, USA) was performed on a 7500 FAST Real-Time PCR System (Applied Biosystems). The relative expression levels of the mRNA were calculated and quantified using the 2−ΔΔT method after normalization to the expression of the control. GAPDH served as the endogenous control. The primer sequences are described in Additional file 2: Table S2 and were purchased from Comate Bioscience (Institute of Biotechnology, Jilin, China).
Electron microscopy was performed as previously described . Pancreatic cancer cell lines were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide buffer. Tissues were embedded in spur resin, and thin sections were cut. The sectioned grids were stained with a saturated solution of uranyl acetate and lead citrate. Sections were examined at 80 kV using a JEOL 1200EX transmission electron microscope.
The GFP-mRFP-LC3 lentivirus was purchased from GeneChem (Shanghai China). Pancreatic cancer cell lines (CFPAC-1 and PANC-1) cultured on covers lips were transduced with the control and the GFP-mRFP-LC3 lentiviral vectoros and were then selected with puromycin (Sigma-Aldrich, St. Louis, MO, USA) for one week. Stably transfected cells were infected with the LV-UBL4A-Flag, LV-shUBL4A, LAMP1 plasmid, si-LAMP1 and their respective controls. The cells were viewed under a fluorescence microscope. Theoretically, GFP is a stably folded protein and is relatively resistant to lysosomal proteases. However, the low pH level inside the lysosomes quenches the fluorescent signal of the GFP. Therefore, autophagosomes and autolysosomes were labeled yellow (mRFP and GFP) and red (mRFP only), respectively. The numbers of GFP and mRFP dots were determined by manual counting of fluorescent puncta in five high-power fields (20×, Olympus) and analyzed using Image-Pro Plus v6.0 software. We calculated the ratio of autolysosomes (red) to autophagosomes (yellow) per cell to evaluate the extent of autophagosomes maturation into autolysosomes.
The tumor cells transfected with Lv-UBL4A-Flag and Lv-shUBL4A as well as their controls were seeded on 24-well plates. The cells were fixed with 4% paraformaldehyde for 30 min and were permeabilized with 0.5% Triton X-100 for 20 min. After incubation for 2 h with anti-UBL4A (Proteintech, Wuhan, Hubei, China), anti-LAMP1 (Abcam, Shanghai, China) and anti-LC3B (Cell Signaling Technology, Danvers, MA, USA), the cells were washed with PBS three times. Then, the cells were incubated with secondary antibodies for 1 h (Bioss, Beijing, China), and 4′6-diamino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China) was added to stain the cell nuclei. Finally, the cells were viewed with a fluorescence microscope (20×, Olympus). Information on the primary antibodies for immunofluorescence is provided in Additional file 3: Table S3.
Lyso-tracker red staining
Transfected cells were stained with Lyso-Tracker Red (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions, and a fluorescence microscope (20×, Olympus) was used to observe fluorescence. The red signals (lysosome) were quantified and are shown in s bar graph with relevant statistics.
First, 2 × 107 cells were lysed with 500 μL of ice-cold polysome lysis buffer (100 mM KCl; 4 mM MgCl2; 10 mM HEPES, pH 7.0; 0.5% Nonidet P-40; 1 mM DTT; 100 units/ml RNase OUT; and 40 μL/mL complete protease inhibitor cocktail) for 10 min on ice. Then, the cell lysates were collected after centrifugation. UBL4A antibodies (Proteintech, Wuhan, Hubei, China) or control immunoglobulin (IgG) (Beyotime Biotechnology, Shanghai, China) were added to protein G agarose beads (EMD Millipore Corporation, Temecula, CA, USA) and allowed to bind while rotating at 4 °C overnight (for UBL4A and LAMP1) or 2 h (for IgG). The lysates were precleared with an IgG antibody and then incubated with precoated beads for 2 h at RT on a rotator. After the beads were washed, the complexes were boiled for 10 min at 100 °C and loaded on a gel for western blotting. Information on the primary antibodies for co-IP is provided in Additional file 3: Table S3.
Orthotopic tumor model
Orthotopic tumor models were created as previously described . The study protocol was approved by the Institutional Review Board of The First Affiliated Hospital of Harbin Medical University. Briefly, two luciferase-expressing cell lines (CFPAC-1-UBL4A and PANC-1-shUBL4A and their control cells) (5 × 106/0.2 mL) were injected into the right flanks of nude mice. Then, 1 mm3 pieces of tumor harvested from four mice were transplanted into four groups of mouse pancreatic tails. The animals were imaged weekly using the Night OWL II LB983 imaging system in vivo (Berthold Technologies GmbH & Co. KG, Germany). After 5 weeks of xenograft procedures, the mice were sacrificed, and the numbers of visible metastatic lesions in the gut, mesentery, liver, spleen and kidneys were recorded. The primary and metastatic pancreatic tumors were excised, weighed, and fixed in 4% paraformaldehyde.
Statistical analysis was performed with SPSS 19.0 software or GraphPad Prism 6.01 software. The data were shown as the mean ± standard deviation (SD). Pearson analysis and Kaplan–Meier survival analysis were used to evaluate the statistical significance, and the variance between the two groups was analyzed using Student t-tests. Differences were considered significant when *, P < 0.05; **, P < 0.01; ***, P < 0.001; and non-significant when P > 0.05.
Details on other experimental procedures are described in the Supplementary Methods (Additional file 9: Supplementary Methods).
UBL4A expression decreases in PDAC, and high UBL4A expression is correlated with longer survival
UBL4A inhibits tumor proliferation and metastasis in PDAC cell lines
UBL4A suppresses tumor proliferation and metastasis through inhibition of autophagy
To explore whether accelerated autophagosome synthesis or reduced autophagic vacuole maturation and degradation is responsible for UBL4A-induced autophagosome accumulation in cells, the autophagy-related genes ATG5 and ATG7, key molecules involved in autophagosome formation, were introduced [26, 27]. Western blot assays were subsequently used to analyze the expression levels of ATG5 and ATG7 among different groups. However, as shown in Additional file 7: Figure S4a, neither of these two ATGs exhibited noteworthy changes with the downregulation or upregulation of UBL4A. Thus, we speculate that UBL4A may play a role in autolysosome degradation instead of autophagosome formation. Consistently, our data showed that CQ abolished the effects of UBL4A on autophagy inhibition and tumor suppression and had no synergetic function with UBL4A (Fig. 3c-j, Additional file 6: Figure S3). These findings confirmed that UBL4A participated in regulating autolysosome degradation, which occurs during the late stage of autophagy. In addition, a tandem-labeled GFP-mRFP-LC3 reporter was used to measure autophagic flux. GFP is a stably folded protein that is resistant to lysosomal proteases. The low pH level inside lysosomes quenches the fluorescent signal of GFP. Autophagosomes and autolysosomes were labeled yellow (mRFP and GFP) and red (mRFP only), respectively . As shown in Fig. 3k and m, the ratios of red dots (autolysosomes) to yellow dots (autophagosomes) per cell were significantly decreased in the UBL4A overexpressing groups compared with those in the control groups, indicating that autophagic flux was hampered after UBL4A upregulation. In contrast, suppression of UBL4A increased the ratio of red dots (autolysosomes) to yellow dots (autophagosomes) per cell in comparison with the control groups (Fig. 3l-m). Furthermore, CQ simultaneously blocked autophagic flux and eliminated the influence of UBL4A on autophagic flux (Fig. 3k-m). Together, these data indicate that UBL4A is a potent autophagic inhibitor that causes autophagosome accumulation due to impaired autophagic degradation and suppresses pancreatic cancer proliferation and metastasis by inhibiting autophagy.
UBL4A causes lysosomal dysfunction in PDAC cells
UBL4A directly targets LAMP1 in pancreatic cancer
LAMP1 is involved in UBL4A-mediated inhibition of autophagy and antitumor effects
UBL4A inhibits tumor proliferation and metastasis in an orthotopic tumor model
Autophagy is a dynamic and continuous process involving the formation of autophagosomes (early stage) and lysosomal degradation after fusion (late stage . Autophagy is regarded as a double-edged sword , and its role in cancer is context-dependent and tumor stage-dependent . J Nassour et al. suggested that autophagy is an integral component of the tumor suppressive crisis mechanism and that loss of autophagy is required for the initiation of cancer . However, our previous study showed that elevated autophagy was positively associated with tumor progression in PDAC tissues and cell lines . During the early stages of cancer, autophagy may inhibit tumor initiation by restricting tumor necrosis and inflammatory cell infiltration. However, autophagy tends to act as a promoter of metastasis by enhancing metastatic cell survival and colonization in distant sites during the advanced stages of cancer. Our study showed that an elevated autophagic level was associated with high proliferation and metastasis of PDAC and that CQ exerted a prominent antitumor effect in pancreatic cancer (Fig. 3). Due to the understanding of the prominent role of autophagy in late-stage carcinogenesis, autophagy inhibition has emerged as an appealing therapeutic strategy in pancreatic cancer [39, 40].
UBL4A is a small ubiquitin-like protein encoded by a housekeeping gene . As part of a cytosolic protein quality control complex, the BAG6/BAT3 complex, UBL4A is an essential protein that functions in the protein degradation of defective polypeptides and tail-anchored transmembrane protein biogenesis and delivery [5, 41]. In addition, UBL4A represses tumorigenesis through dephosphorylation of STAT3  and is involved in DNA-damage-induced cell death . Previous studies demonstrated that UBL4A is critical for the migration and the innate immune responses of fibroblasts and macrophages [43, 44]. Moreover, UBL4A interacted with actin-related protein (Arp2/3) and promoted actin branches, which served as “bridges” that guide Akt to the plasma membrane for activation . All these previous studies indicate that this protein is versatile. However, the underlying molecular mechanisms by which UBL4A regulates autophagy and contributes to pancreatic cancer development and progression remain unknown.
In this study, we observed that UBL4A inhibited tumor proliferation and metastasis through the suppression of autophagy. To explore the mechanisms of UBL4A, we showed that overexpression of UBL4A promoted the accumulation of autophagosomes, possibly due to either accelerated autophagosome synthesis or impaired autophagic vacuole maturation and degradation. The autophagy-related genes ATG5 and ATG7 were initially detected. Both of these genes appear to be specifically involved in autophagosome formation . Our study revealed that UBL4A-mediated autophagy inhibition and autophagosome accumulation are not required for accelerated autophagosome synthesis. As a result, we speculated that UBL4A impaired autophagy during the late stage. Next, CQ, which blocks late-stage autophagy, was used to examine the effects of UBL4A on autophagosome processes and the relationship between autophagy and tumor progression. Intriguingly, CQ eliminated the influence of UBL4A on autophagic flux but could not cooperate with UBL4A during the inhibition of tumor proliferation and metastasis, which indicated that UBL4A was a potent autophagic inhibitor that caused impaired autophagic degradation. We also found that UBL4A caused lysosomal dysfunction rather than impaired fusion between autophagosomes and lysosomes by verifying the collocation of LC3B and LAMP1. Functional lysosomes have the unique feature of having a highly acidic pH, and lysosomal degradation depends on the concentration and activity of the hydrolases, such as cathepsin B . Previous studies have shown that lysosomotropic compounds exert a suppressive effect downstream of autophagosome formation by inhibiting autophagosome and lysosome fusion and/or blocking the degradation of the autophagic cargo inside autolysosomes [48, 49]. Similarly, our study describes an exact molecular mechanism that regulates late-stage autophagy, particularly lysosomal degradation, which may represent a useful adjuvant in antitumor therapy.
After confirming the role of UBL4A in autophagy inhibition, we further demonstrated that LAMP1 was a direct target of UBL4A. LAMP1 is distributed among autophagic and endolysosomal organelles and is routinely used as a lysosomal marker, and LAMP1-positive organelles are often referred to as lysosomal compartments. Despite its abundance, the role of LAMP1 in autophagy is ambiguous and insignificant according to previous studies . Recently, Cheng et al. demonstrated that a significant portion of LAMP1-labeled organelles lack major lysosomal hydrolases. Their data called for caution in interpretation: LAMP1-labeled organelles do not necessarily represent degradative lysosomes or autolysosomes [50, 51]. Similarly, our study demonstrated that the restoration of LAMP1 abolished UBL4A-knockdown induced autophagy activation and influenced the expression and maturation of lysosomal hydrolases. Although the exact molecular mechanism is not clear, LAMP1 actually had an impact on regulating the expression and activity of lysosomal hydrolases, at least in UBL4A-mediated inhibition of autophagy. LAMP2, which is 37% identical to LAMP1, is essential for the degradation of the autophagosomal content via the proper fusion of lysosomes with autophagosomes in the last stage of autophagic flux . To explore whether LAMP2 participates in UBL4A-mediated inhibition of autophagy, we investigated the expression of LAMP2 in UBL4A downregulated and upregulated groups by western blotting. Our results provide no evidence that UBL4A could regulate LAMP2, at least in pancreatic cancer (Additional file 8: Figure S5e). Together, the above results comprehensively demonstrate that LAMP1 plays a crucial role in UBL4A-induced autophagy inhibition, and they provide novel insights regarding LAMP1 in autophagy regulation.
Our findings suggest a new mechanism for the regulation of proliferation and metastasis by the UBL4A/LAMP1/autophagy axis in pancreatic cancer and reveal a direct interaction between UBL4A and LAMP1. However, in this study, we did not determine whether UBL4A regulates the expression of LAMP1through an exact mechanism, such as the ubiquitination–proteasome pathway, or which signaling pathways contribute to the UBL4A-induced antitumor effects in pancreatic cancers. Nevertheless, our data indicate that UBL4A suppresses pancreatic cancer development by directly regulating LAMP1.
The authors would like to thank Dr. Yue Yuan from The First Affiliated Hospital of Harbin Medical University for her selfless assistance.
HZC and LL designed this research. HZC performed most experiments in this work and drafted this manuscript. LL, JSH and LJ helped with reviewing this manuscript for clarity. JSH helped with the collection of clinical specimens. ZJZ and CDC helped with the animal experiment. YLL helped with the western bolt experiments. GQZ and TZ helped with the PCR experiments. SHP helped with the IHC. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (81372613, 81670583 and 81800572), The China Postdoctoral Science Foundation (2017 M621300, 2018 T110316), The Heilongjiang Providence Postdoctoral Science Foundation (LBH-Z17179, LBH-TZ17).
Ethics approval and consent to participate
PDAC and normal pancreatic tissues were acquired from patients undergoing a surgical procedure at The First Affiliated Hospital of Harbin Medical University (Harbin, China). Patient consent was obtained prior to the initiation of the study.
Consent for publication
We have obtained consents to publish this paper from all the participants of this study.
The authors declare that they have no competing interests.
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