PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2
Authors Liu X, Li C, Fu Y, Liu J
Received 5 November 2019
Accepted for publication 24 December 2019
Published 14 January 2020 Volume 2020:13 Pages 401—411
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Sanjeev Srivastava
Xiao Liu, Chengyuan Li, Yunfeng Fu, Jing Liu
Department of Hematology, The Third Xiangya Hospital of Central South University, Changsha 410013, People’s Republic of China
Correspondence: Jing Liu; Yunfeng Fu
The Third Xiangya Hospital of Central South University, Tongzipo Road 138, Changsha 410013, People’s Republic of China
Tel/Fax + 86-073188618511
Email [email protected]; [email protected]
Introduction: Treatment of bortezomib (BTZ) improves the clinical outcomes of patients with multiple myeloma (MM). However, primary resistance and acquired resistance to BTZ frequently develop in patients with MM. PH domain leucine-rich repeat protein phosphatase (PHLPP) plays an important role in chemoresistance in a number of cancers. However, the role of PHLPP on MM remains unclear. In this study, we investigated the role of PHLPP in BTZ-resistant MM cells.
Methods: BrdU assays, immunoprecipitation, flow cytometry analyses, and immunofluorescence assays were performed.
Results: PHLPP and lysosome-associated membrane protein 2 (LAMP2) levels were downregulated in BTZ-resistant MM cells compared with BTZ-sensitive MM cells, accompanied by inactivation of autophagy pathway evaluated by a reduction in Beclin1, Atg5 and LC3B and increase in p62. Gain- and loss-of-function experiments revealed that PHLPP partially re-sensitized MM cells to BTZ. In addition, PHLPP overexpression increased whereas PHLPP knockdown reduced LAMP2 expression, subsequently regulating the autophagy pathway in MM cells. Further findings demonstrated that LAMP2 knockdown reversed PHLPP-mediated cell apoptosis and autophagy activation in MM cells.
Conclusion: This study demonstrated that PHLPP is a potential strategy for overcoming BTZ resistance in patients with MM.
Keywords: multiple myeloma, PHLPP, bortezomib, resistance, chaperone-mediated autophagy, LAMP2
Multiple myeloma (MM) is a common hematologic malignancy of plasma cells (accounting for ~1% of hematologic malignancy).1 Recently, treatments with immunomodulatory drugs, monoclonal antibodies, and second-generation proteasome inhibitors, such as bortezomib (BTZ), have increased recurrence-free survival and overall survival rates for patients with MM.2,3 However, most patients who have primary or acquired resistance to BTZ will suffer a relapse.4
PH domain leucine-rich repeat protein phosphatase (PHLPP) is a Ser/Thr protein phosphatase that plays a critical role in maintaining cellular homeostasis by directly regulating Akt, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK).5 Loss of PHLPP expression is associated with tumor progression, and increasing evidence indicates that PHLPP is a tumor suppressor and a potential therapeutic target.6 Patients with lung adenocarcinoma that have low PHLPP levels have poorer survival rates than those with high PHLPP.7 In addition, hypoxia-induced chemoresistance is associated with the downregulation of PHLPP in colon cancer cells,8 suggesting that PHLPP plays an important role in chemoresistance. However, the role of PHLPP on MM remains unclear.
Lysosomal associated membrane proteins (LAMPs) family is characterized by an evolutionary-conserved membrane-proximal LAMP domain and composed of five known members: LAMP1, LAMP2, LAMP3, LAMP4 and LAMP5.9 LAMP2 has been well documented in a variety of cellular processes including autophagy, specifically chaperone-mediated autophagy (CMA), a process that targets specific proteins to degradation by lysosomes.10 LAMP2 can affect the lysosomes localization and the autophagic flux, and LAMP2 deficiency was responsible for Azacytidine resistance in MDS/AML cells.11 However, the expression and roles of LAMP2 in BTZ-resistant MM cells are unknown.
In this study, we found that PHLPP levels were decreased in bortezomib-resistant cells and bone marrow samples of patients with MM, and overexpression of PHLPP partially sensitized MM cells to BTZ by increasing LAMP2. Our study indicates that the rescue of PHLPP is a potential strategy for the management of acquired BTZ resistance in multiple myeloma.
Materials and Methods
Clinical Samples Collection
Three newly diagnosed with MM and three BTZ-resistant MM patients were enrolled in this study. This study was approved by the Ethics Committee of the Third Xiangya Hospital of Central South University. Written informed consent was obtained from each subject in accordance with the Declaration of Helsinki. Bone marrow tissues were obtained from these patients. Cell flow cytometry was used to sort CD138+ cells.
The MM cell line U266 was purchased from Cell Bank (Chinese Academy of Sciences, Beijing, China). Cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium (GlutaMAX™-I, cat no. 72400120, Gibco). 10% fetal bovine serum (Gibco, CA, USA) was added in the medium. Cells were cultured under the condition in a 37°C humidified atmosphere of 5% CO2.
Generation of BTZ-Resistant U266 Cells
The bortezomib-resistant U266 cells (U266-R) were established as follows: U266 cells were treated with the initial concentration of bortezomib (2 nM). The medium was refreshed once every 2 days. After cultured for 2 weeks under the same concentration, the dose was increased for two folds, and continuously maintained for 2 weeks. The cycles were repeated 10 times, so that the U266-R cells can be resistant to bortezomib up to 20 nM. The cells were kept under 10 nM bortezomib pressure before experimental usage.
Cell Transfection and Treatment
Lentivirus mediated PHLPP (shRNA) and LAMP2 (shRNA) downregulation, and PHLPP and LAMP2 overexpression were obtained from Genefullen (Guangzhou, China). The lentivirus-containing scramble sequences were used as control. Lentivirus infection was performed according to the manufacture’s protocol. The cells were then co-treated with rapamycin (RAP) or hydroxychloroquine (HCQ), or BTZ (5 nM or 40 nM) for 48 hrs and then used for further analysis.
BrdU Cell Proliferation ELISA kit (Abcam, Shanghai, China) was used in this experiment. After indicated treatment, the cells were added BrdU and incubate for 12 hrs to incorporate BrdU into their DNA. The cells then were fixed with Fixing Solution and incubate at room temperature. Add the TMB Solution to each well for 5 mins and then add the Stop Solution to stop the reaction. The color development was recorded immediately.
MEBCYTO Apoptosis Kit (MBL Beijing Biotech Co., Ltd, Beijing, China) was used in this experiment. After treatment, cells were collected and then resuspended in binding buffer. Annexin V-FITC (5 μL) and Propidium Iodide (5 μL) were added and mixed well. The cells were incubated with the mixed solution for 15 mins. Finally, flow cytometric analysis was performed in Attune NxT (Thermofisher, Shanghai, China).
Protein was extracted using RIPA (Beyotime, Hangzhou, China) and their concentrations were determined by Enhanced BCA Protein Assay Kit (Beyotime, Hangzhou, China). Sixty microgram proteins were separated in 10% SDS/PAG and transferred on PVDF membrane. The membranes were incubated with primary antibody: anti-LAMP2 antibody (1:1000, cat no. 49067, Cell Signaling Technology), anti-LAMP1 antibody (1:1000, cat no. ab13523, Abcam), anti-LAMP2A antibody (1:1000, cat no. ab125068, Abcam), anti-LAMP2B antibody (1:1000, cat no. ab18529, Abcam), anti-AKT antibody (1:1000, cat no. ab18785, Abcam), anti-phospho-Akt (Ser473) antibody (1:1000, cat no. 4058, Cell Signaling Technology), anti-PHLPP antibody (1:1000, cat no. ab62830, Abcam); anti-Beclin1 antibody (1:1000, cat no. ab210498, Abcam); anti-Atg5 antibody (1:1000, cat no. ab228668, Abcam); anti-p62 antibody (1:1000, cat no. ab56416, Abcam); anti-LC3B antibody (1:1000, cat no. ab51520, Abcam). GAPDH (1:1000, cat no. ab181602, Abcam) was used as control. After being washed, the membranes were reacted with peroxidase-conjugated secondary antibody. The signals were visualized by enhanced chemiluminescence reagents (GE, Fairfield, CT, USA).
Immunoprecipitation was performed to determine the interaction between PHLPP and LAMP2. Ice-cold cell lysis buffer (500 μL) was added to each plate and incubate for 5 mins. The cells were collected to microcentrifuge tubes. The samples were sonicated on ice three times for 5 s each and micro-centrifuged for 10 mins at 14000 g, 4°C. 200 μL cell lysate was incubated with PHLPP or LAMP2 primary antibodies with rocking overnight at 4°C. Protein A agarose beads (Beyotime, Hangzhou, China) were added and incubated with for 3 hrs at 4°C. After microcentrifuge, the pellets were washed with cell lysis buffer and were resuspended with SDS sample buffer and heated to 98°C for 5 mins. 30 μL of the sample was loaded on SDS-PAGE gel (12%) for Western blotting analysis.
After blocked with 5% BSA, the cells were incubated with primary antibodies (anti-LAMP2 antibody, 1:200 and anti-PHLPP, 1:500) at 4°C overnight. The cells were incubated with appropriate secondary antibodies at 37°C for 1 hr. The cells were stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (1:1000, Beyotime, Hangzhou, China) for 5 mins and mounted on slides. Immunofluorescence images were captured by a fluorescence microscope (BX51, Olympus, Japan).
Lysosome was stained with Lysotracker-Red DND-99 (Molecular Probes, Eugene, USA). After infected with PHLPP lentivirus (labelled with EGFP), U266 cells were incubated in the presence of 50 nM Lysotracker-Red for 30 mins at 37°C. Immunofluorescence images were captured by a fluorescence microscope (BX51, Olympus, Japan).
Animal experiments were approved by the Ethics Committee for Animal Research of the Third Xiangya Hospital of Central South University following the National Research Council’s Guide for the Care and Use of Laboratory Animals (Eight Edition). The male, 2-month-old nude mice were purchased from Animal Center of Central South University. U266 were transfected with PHLPP or negative control for 48 hrs. Transfected U266 cells (1 × 106) were then subcutaneously injected into nude mice (N = 5/each group). Tumor volume was calculated as 0.5 × L × W,2 where L and W are long and short diameters of the tumor, respectively.
GraphPad Prism software (GraphPad Software Inc., La Jolla, CA) was used for statistical analyses. Student’s t-test for two-group and one-way ANOVA with post hoc Bonferroni test for three or more groups were used to assess the statistical significance. P < 0.05 was considered statistically significant.
PHLPP and LAMP2 Levels are Increased in BTZ-Resistant MM Cells
To investigate the role of PHLPP and LAMP2 in BTZ-resistant MM, we measured their expression levels in plasma cell from patients with MM and BTZ-resistant MM cell lines. We found that LAMP2 and PHLPP expression levels in BTZ-resistant plasma cells of human samples were downregulated compared with BTZ-sensitive samples (Figure 1A). Their expressions were also lower in BTZ-resistant U266 cells compared with parental U266 cells (Figure 1B). However, the expression of another member of LAMP family, LAMP1 was comparable between BTZ-sensitive and BTZ-resistant patients with MM. The expression of LAMP2 isoform LAMP2A, not LAMP2B, was significantly downregulated in BTZ-resistant patients with MM compared the BTZ-sensitive patients with MM (supplementary Figure 1A). In addition, downregulation of PHLPP and LAMP2 was accompanied by inactivation of autophagy, as determined by decreased Beclin1 and Atg5 levels, and ratio of LC3B-II/LC3B-I, and increased p62 expression in plasma cells from BTZ-resistant MM and U266-R cells (Figure 1). These results suggest that PHLPP downregulation is associated with LAMP2 and chaperone-mediated autophagy and involved in BTZ resistance.
PHLPP Sensitizes MM Cells to BTZ
PHLPP was knocked-down in U266 cells and was overexpressed in U266-R cells (Figure 2A). PHLPP knockdown significantly promoted U266 cell proliferation, and inhibited cell apoptosis following BTZ treatment (Figure 2B and C). However, PHLPP overexpression significantly inhibited U266-R cell proliferation, and induced cell apoptosis following BTZ treatment (Figure 2B and C). These results suggest that PHLPP sensitizes MM cells to BTZ treatment.
PHLPP Suppresses MM Cells Growth in vivo
Furthermore, we performed xenografted tumor experiments in nude mice using PHLPP-expressing U266 cells to examine the effects of PHLPP on tumor growth in vivo. PHLPP overexpression slowed down tumor growth in vivo (Figure 2D). Immunohistochemical staining showed that PHLPP and LAMP2 expression were upregulated in tumor tissues (Figure 2E).
PHLPP Interacts with LAMP2
Given that PHLPP expression was associated with LAMP2 expression, we investigated whether PHLPP interacts physically with LAMP2. Immunofluorescence assays showed that PHLPP and LAMP2 were co-localized in U266 cells (Figure 3A). Co-immunoprecipitation (co-IP) experiments further confirmed that PHLPP interacts with LAMP2 (Figure 3B), and they were co-expressed in the lysosome (Figure 3C). In addition, we found that knockdown of PHLPP decreased LAMP2 expression (Figure 3D). Knockdown of PHLPP also reduced Beclin1 and Atg5 levels and ratio of LC3B-II/LC3B-I, and increased p-AKT(ser473) and p62 expression, suggesting autophagy signaling inactivation in U266 cells, whereas overexpression of PHLPP increased the expression of LAMP2 and LAMP2A, but did not alter the expression of LAMP1 and LAMP2B (supplementary Figure 1B) and inhibited phosphorylation of AKT, activating autophagy signaling in U266-R cells (Figure 3D).
PHLPP Partially Sensitizes MM Cells to BTZ Through LAMP2 and Autophagy
We next tested the role of LAMP2 in BTZ-induced cell apoptosis. We found that LAMP2 overexpression enhanced while LAMP2 knockdown attenuated BTZ-induced growth inhibition and cell apoptosis (supplementary Figure 2). To investigate the role of LAMP2 in PHLPP-mediated BTZ sensitization, LAMP2 was knocked down by shRNA in U266-R cells (Figure 4A) and overexpressed in U266 cells (Figure 4B). Under BTZ treatment, LAMP2 knockdown reversed PHLPP-mediated autophagy activation as determined by downregulation of Beclin1 and Atg5 levels and the ratio of LC3B-II/LC3B-I and upregulation of p-AKT(ser473) and p62 (Figure 4A), proliferation inhibition (Figure 4C), and apoptosis (Figure 4D) in U266-R cells. LAMP2 overexpression rescued the effects of shPHLPP treatment on autophagy (Figure 4B), cell proliferation (Figure 4E), and cell apoptosis (Figure 4F) in U266 cells, suggesting that LAMP2 is required for PHLPP in re-sensitizing MM cells to BTZ. Moreover, pharmacological activator of autophagy RAP treatment significantly decreased cell proliferation and enhanced cell apoptosis compared with shPHLPP transfection alone in U266 cells (Figure 5A and B); the autophagy inhibitor hydroxychloroquine (HCQ) treatment dramatically promoted cell proliferation and reduced cell apoptosis compared with PHLPP transfection alone in U266-R cells (Figure 5C and D). These results suggest that PHLPP sensitizes MM cells to BTZ through LAMP2 and the autophagy pathway.
In this study, we found that PHLPP and LAPM2 were downregulated in BTZ-resistant MM cells. Induced PHLPP expression partially re-sensitized MM cells to BTZ through activating the autophagy pathway.
MM is a common hematologic malignancy, constituting 1% of all malignancies. Although treatment regimens have greatly improved, MM remains incurable because of resistance to drugs such as dexamethasone and bortezomib.12 Drug resistance involves the dysregulation of various signaling pathways, including the JAK/STAT, MEK/MAPK, and PI3K/AKT.13 AKT phosphorylation activates AKT signaling, which is associated with anti-apoptotic effects of plasma cell-derived growth factor against the effects of dexamethasone.14 MM cells produce the growth factor IGF-1, and high levels of IGF1 and its receptor (IGF-1R) are associated with poor survival and BTZ resistance in patients with MM.15 The IGF-1R inhibitor OSI-906 can increase the cytotoxicity of BTZ in MM cells, and PI3K and AKT are the downstream targets of IGF1.16 AKT inhibition also causes BTZ-resistant cell death.17 Therefore, combination bortezomib and OSI-906 therapy could be a useful treatment for patients with BTZ resistance.
PHLPP negatively regulates AKT activity, and FK506-binding protein 51 functions as a scaffolding protein for Akt and PHLPP. PHLPP can promote dephosphorylation of AKT and enhances the efficacy of chemotherapy.18 PHLPP overexpression inactivates Akt, whereas PHLPP knockdown increases phosphorylation and activation of Akt.19 In addition, hypoxia-induced chemoresistance is associated with the downregulation of PHLPP in colon cancer cells.8 In this study, we found that PHLPP levels were reduced in BTZ-resistant MM cells, and induced PHLPP expression re-sensitized MM cells to BTZ. Interestingly, we found that PHLPP inhibited proliferation and promoted apoptosis in MM cells by enhancing CMA. CMA contributes to the maintenance of proteostasis through the degradation of cytosolic proteins in lysosomes.20 Cytosolic chaperones delivered CMA substrates to the lysosomal surface, and are internalized through a membrane translocation complex.21 It was previously found that PHLPP positively regulated CMA through inhibition of lysosomal Akt.22 However, in addition to AKT and autophagy pathways, PHLPP is also associated with activation of NF-κB signaling.23 Tang et al reported that PHLPP upregulation was mediated by NF-κB transcriptional activity.24 The NF-κB pathway regulates numerous genes, which influence the development and pathogenesis of multiple myeloma. IGF-1 was reported to be important for cell survival by AKT signaling pathways, which further mediate NF-κB activation in MM cells.25 Thus, there may be a negative feedback loop among NF-κB, PHLPP and Akt, which may lead to a mild effect on cell apoptosis. However, the precise mechanism needs more evidences to illustrate.
Low expression of LAMP2 was associated with poor overall survival in a cohort of 150 AML patients. Moreover, in Azacytidine (Aza)-treated MDS cell lines and cells from MDS/AML patients, a progressive reduction in LAMP2 expression was correlated with a loss of sensitivity to Aza.11 Bao et al found that the downregulation of LAMP2 in the lung cancer cells increased their resistance to Temozolomide.26 In LAMP1/LAMP2 double-knockout mice, autophagic vacuoles accumulated in many tissues suggesting impaired autophagosome maturation.27 In this study, we found that LAMP2 but not LAMP1 was downregulated in BTZ-resistant patients with MM and the expression of LAMP2A, not LAMP2B, was significantly downregulated in BTZ-resistant patients with MM compared the BTZ-sensitive patients with MM. LAMP2A was encoded by transcript variant A of Lamp2 gene, which is the longest and predominant form of this gene. These results indicated that LAMP2A is the executor in the regulation of CMA in MM cells. Re-expression of LAMP2 in LAMP2-deficient BTZ-resistant MM cells restored chaperone-mediated autophagy (CMA), highlighting the key role of LAMP2 in this process. CMA is a highly selective form of autophagy that allows the elimination of substrates endowed with a KFERQ motif through direct transport to the lysosome. Lysosomes can sensitize cancer cells to lysosomal membrane permeabilization and cell death.28,29 In this study, we found that LAMP2 levels were reduced in BTZ-resistant MM cells, and knockdown of LAMP2 reversed PHLPP-mediated cell apoptosis in BTZ-resistant U266 cells. Therefore, LAMP2 contributes to BTZ resistance and cell apoptosis may be via CMA activation. Our results showed that PHLPP interacted with LAMP2, and LAMP2 is required for PHLPP-mediated autophagy. It would be reasonable that PHLPP acts primarily to interact with and stabilize LAMP2 on the lysosomes to promote the fusion between autophagosome and lysosome (ie, formation of autolysosome) and thereby removal of autophagosome.
In summary, our findings reveal for the first time that interaction of PHLPP and LAMP2 partially sensitizes MM cells to BTZ by enhancing autophagy. Rescue of PHLPP is a potential strategy for the management of BTZ resistance in patients with MM.
Ethics Approval and Informed Consent
This study was approved by the Ethics Committee of The Third Xiangya Hospital, Central South University. Informed consent was obtained from each subject in accordance with the Declaration of Helsinki. Animal experiments were approved by the Ethics Committee for Animal Research of the Third Xiangya Hospital of Central South University following the National Research Council’s Guide for the Care and Use of Laboratory Animals (Eight Edition).
Authors would like to thank Dr Yanwei Luo for his help in collecting some of the data for this study.
All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
The authors declare that they have no conflicts of interest in this work.
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