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A novel role of lysophosphatidic acid (LPA) in human myeloma resistance to proteasome inhibitors
Journal of Hematology & Oncology volume 15, Article number: 55 (2022)
Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that regulates cell proliferation, survival, and migration. However, its role on human multiple myeloma (MM) cells is largely unknown. In this study, we show that LPA, which is highly elevated in MM patients, plays an important role in protecting human MM cells against proteasome inhibitor (PI)-induced apoptosis. LPA bound to its receptor LPAR2 activated its downstream MEK1/2-ERK1/2 signaling pathway and enhanced oxidative phosphorylation (OXPHOS) in mitochondria in MM cells. Increased OXPHOS activity produced more NAD+ and ATP, reduced proteasome activity, and enhanced protein folding and refolding in endoplasmic reticulum (ER), leading to induction of MM resistance to PIs. Importantly, inhibiting LPAR2 activity or knocking out LPAR2 in MM cells significantly enhanced MM sensitivity to PI-induced apoptosis in vitro and in vivo. Interestingly, primary MM cells from LPA-high patients were more resistant to PI-induced apoptosis than MM cells from LPA-low patients. Thus, our study indicates that LPA-LPAR2-mediated signaling pathways play an important role in MM sensitivity to PIs and targeting LPA or LPAR2 may potentially be used to (re)sensitize patients to PI-based therapy.
To the Editor,
Multiple myeloma (MM) is a hematological malignancy that remains largely incurable and most patients relapse after one or more treatment regimens [1, 2]. The therapeutics currently available improve patient survival and quality of life, but resistance to therapy and disease progression remain unsolved issues . Proteasome inhibitors (PIs) have been used as the frontline therapies for newly diagnosed and relapsed or refractory MM patients for the last two decades [4, 5]. Although PIs have shown encouraging therapeutic results, primary and secondary drug resistances and relapse after long-term treatment are inevitable in most treated patients. LPA is a naturally occurring phospholipid that regulates cell proliferation , survival  and migration  and exerts its effects on target cells by binding to G protein-coupled receptors (GPCRs), including LPAR1-6 . Herein, we explored the mechanism underlying the regulation of LPA/LPAR2 axis on MM resistance to PI-induced apoptosis in vitro and in vivo.
MM patients (Fig. 1a) and MM-bearing mice (Fig. 1b) produced high levels of circulating LPA than their healthy controls. Moreover, primary MM cells from LPA-high patients were more resistant to PI-induced apoptosis than MM cells from LPA-low patients (Fig. 1c), and LPA treatment significantly decreased apoptosis of human primary MM cells (Fig. 1d) and cell lines (Additional file 1: Fig. S1a) induced by bortezomib (BTZ) or carfilzomib (CFZ), but not melphalan, dexamethasone, or pomalidomide (Additional file 1: Fig. S1b). Consistent with MM data in Oncomine (Fig. 1e–f), only LPAR2 among 6 LPA receptors was highly expressed in human primary MM cells (Fig. 1g) and cell lines (Additional file 1: Fig. S1c). These results suggested that the effect of LPA on MM may be derived by LPAR2. Indeed, inhibiting LPAR2 activity (Additional file 1: Fig. S1d) or knocking out LPAR2 (Fig. 1h) abolished LPA-induced apoptosis resistance in BTZ- or CFZ-treated MM cells. Further mechanistic study showed that LPA enhanced the phosphorylation of MEK1/2 and ERK1/2 only in Ctr- but not in LPAR2-KO MM cells (Fig. 1i, j, Additional file 1: Fig. S1e–S1f), and inhibiting MEK1/2-ERK1/2 signal cascade significantly attenuated the protective effect of LPA on BTZ- or CFZ-induced apoptosis in MM cells (Fig. 1k, Additional file 1: Fig. S1g).
To elucidate the molecular mechanisms downstream of LPA-LPAR2-MEK1/2-ERK1/2 pathway, Ingenuity Pathway Analysis (IPA) (Fig. 1l) and gene set enrichment analysis (GSEA) (Fig. 1m, Additional file 1: Fig. S2a–S2b) were used. Results showed that LPA significantly increased mitochondrial oxygen consumption rates (OCRs) in MM cells (Additional file 1: Fig. S2c–S2d) and this increase was impaired when LPAR2 was absent (Fig. 1n–o) or MEK1/2-ERK1/2 signal was deficient (Additional file 1: Fig. S2e–S2g). Additionally, mitochondrial respiration inhibited by BTZ or CFZ was restored by LPA treatment (Fig. 1p, Additional file 1: Fig. S2h–S2i) along with increased production of NAD+ (Fig. 1q) and ATP (Fig. 1r) and reduced activity of 26S proteasome (Fig. 1s) due to disruption of NAD+/DADH balance consistent with our GSEA analysis (Additional file 1: Fig. S2j–S2k) and a previous report . As enhanced OXPHOS and ATP production are involved in ER protein folding/refolding essential for MM cell survival , we further analyzed this process in MM cells. Results showed that genes involved in ER protein folding/refolding were highly expressed in MM cells of patients compared to normal plasma cells (Fig. 2a, Additional file 1: Fig. S3a) and positively correlated with the level of LPAR2 in MM patients (Fig. 2b, Additional file 1: Fig. S3b). ER protein folding/refolding ability (Fig. 2c) and ER ATP distribution (Fig. 2d–e) were increased in LPA-treated MM cells. ER retained relatively high ROS levels due to protein folding processes (Additional file 1: Fig. S3c–S3f) and ROS assays (Additional file 2)  showed that the capacity of MM cells to buffer the formation of ROS was enhanced by LPA (Fig. 2f, Additional file 1: Fig. S3g). Suppression of MEK1/2-ERK1/2 signal by PD184352 and SCH772984 abolished this buffer capacity (Fig. 2f, Additional file 1: Fig. S3g), further suggesting the dependence of LPA function on MEK1/2-ERK1/2 pathway.
To further investigate the translational potential of targeting LPAR2-mediated signal cascade to overcome MM cell PI resistance in vivo, we examined the therapeutic effect of CFZ on human Ctr- or LPAR2-KO ARP1- (Fig. 2g, h) or MM.1S-Luc (Fig. 2i, Additional file 1: Fig. S3h) bearing mice. Consistent with in vitro results, CFZ treatment resulted in significantly smaller tumor burdens and prolonged survival in mice bearing LAPR2-KO MM cells compared to Ctr-KO MM cells (Fig. 2g–i, Additional file 1: Fig. S3h). Similarly, combination of CFZ with LPAR2 inhibitor dramatically reduced tumor burden and prolonged mouse survival compared to CFZ alone (Fig. 2j, k), indicating the translational potential of targeting LPAR2-mediated signal cascade to overcome MM cell PI resistance in vivo.
In summary, we described a novel mechanism underlying the induction of MM resistance to PI-induced apoptosis. Our findings may not only contribute to a better understanding of the importance of the bioactive lipid in MM resistance of PIs but also highlight the importance of targeting LPA-LPAR2-mediated signaling pathway as a potential therapeutic approach to overcome MM resistance to PI treatment in patients.
Availability of data and materials
Datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
G protein-coupled receptors
Oxygen consumption rate
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The authors thank Research Core services in the Houston Methodist Research Institute for their support. They also thank Dr. Fumihiko Urano, Division of Endocrinology, Metabolism & Lipid Research, Washington University in St. Louis and Dr. Kohsuke Kanekura, Department of Molecular Pathology, Tokyo Medical University, for kindly gift of the full sequences of MERO-GFP.
This work was supported by Startup Support from Houston Methodist Research Institute, Houston Methodist Hospital and Cancer Prevention & Research Institute of Texas Recruitment of Established Investigator Award (RR180044) and High-Impact/High-Risk Research Award (RP210868). Q.Y. and his research group are also supported by NCI R01s (CA200539, CA211073, CA214811 and CA239255).
Ethics approval and consent to participate
This study was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) and Institutional Review Board (IRB) of Houston Methodist Research Institute (Protocol No. 0918-0053; IRB #Pro00019970). All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996).
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Additional file 1.
Supplementary Figures and Figure legends.
Additional file 2.
Supplementary Materials and Methods.
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Su, P., Xiao, L., Ye, L. et al. A novel role of lysophosphatidic acid (LPA) in human myeloma resistance to proteasome inhibitors. J Hematol Oncol 15, 55 (2022). https://doi.org/10.1186/s13045-022-01269-5
- Multiple myeloma
- Proteasome inhibitor
- Drug resistance