Introduction
Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). It accounts for 10% of all hematological malignancies [
1,
2]. Development of novel pharmaceutical agents has resulted in major advances in the treatment of MM in the last two decades [
3]. Treatment strategies with that combined immune-modulatory drugs, proteasome inhibitors, conventional chemotherapy and monoclonal antibodies resulted in substantial progression in treatment outcome. However, despite improvements in patient survival rates, MM remains an incurable disease. Acquired or de novo resistance to current anti-MM therapy remains a major treatment obstacle [
4,
5]. Novel new therapies are thus in need.
The transient receptor potential (TRP) channel superfamily is one of the largest families of cation channels [
6]. Transient Receptor Potential Vanilloid type 1 (TRPV1) was the first identified and is the most extensively studied member of the vanilloid receptor subfamily of TRP ion channels. The TRPV1 receptor is a non-selective cation channel with a preference for calcium transmission and was initially identified as a specific receptor for capsaicin that causes a burning sensation [
7]. Further studies revealed that TRPV1 is a polymodal receptor and is sensitive to multiple external stimuli that include capsaicin, ethanol, temperature above 43 °C and acid/basic pH changes [
8,
9]. TRPV1 channel is involved in the regulation of calcium signaling, crucial for many cellular processes including proliferation, apoptosis, secretion of cytokines or T cell activation [
10,
11]. Its broad expression in a wide range of tissues such as skin, respiratory airways, gastrointestinal tract, urinary epithelial cells, pancreatic B cells and he immune system, underlines the important role of TRPV1 [
12‐
17]. Currently, TRPV1 was shown to be implicated in neurogenic inflammation, neuropathic pain, autoimmune disorders, immune cells functioning and cancer [
11]. Functional expression of TRPV1 was demonstrated in several human malignancies including breast, prostate, urothelial cancer and glioma [
18‐
22].
The role of TRPV1 channel in MM tumor progression and drug resistance has not been studied. We, therefore, evaluated the role of TRPV1 channel in MM, demonstrating that TRPV1 is expressed in MM cell lines and primary MM cells. TRPV1 inhibition using pharmacological blocker AMG9810 promotes MM cell death and synergizes with anti-MM agent bortezomib. Furthermore, our results reveal the mechanism that underlies the synergism between bortezomib and AMG9810, demonstrating that TRPV1 inhibition disturbs mitochondrial calcium signaling, suppresses bortezomib-induced mitochondrial unfolded protein response (mtUPR) and promotes mitophagy. Altogether, simultaneous TRPV1 and proteasome targeting appears to be promising novel therapeutic strategy in MM.
Materials and methods
Cell lines and MM patient samples
The following human MM cell lines were obtained from ATCC (Rockville, MD, USA): RPMI8226, U266 and NCI-H929. The CAG MM cell line (generated by the group at the University of Arkansas for Medical Sciences (UAMS) [
23]), OPM-1 and OPM-2 (originate from the same individual) were kindly donated by Prof. Israel Vlodavsky, Technion, Israel. Cells were maintained in log-phase growth in RPMI1640 medium (Biological Industries, Israel) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1 mM L-glutamine, 100 U/ml penicillin and 0.01 mg/ml streptomycin (Biological Industries) in a humidified atmosphere of 5% CO
2 at 37 °C. MM cell lines were authenticated in 2019 at the Genomics Center of Biomedical Core Facility, Technion, using the Promega GenePrint 24 System. Primary MM cells were isolated from bone marrow aspirates of myeloma patients. The study was approved by Institutional Review Board of the Sheba Medical Center. Mononuclear cells were collected after separation on Ficoll-Paque (Pharmacia Biotech). MM cells were purified (> 95% purity) by CD138+ isolation using MACS magnetic cell sorter (Miltenyi Biotec Inc.).
Preparation of BMSCs and co-culture experiments
Primary human bone marrow stromal cells (BMSCs) were generated from bone marrow aspirates of consenting healthy donor volunteers. BMSCs were isolated by plate adherence and expanded as previously described [
24].
Inhibitors
The following chemicals were used: bortezomib, carfilzomib, AMG9810, capsaicin and MLN4924 from Cayman.
Cell line transduction
In order to stably over-express CXCR4, RPMI8226 cells were transduced with the lentiviral bicistronic vector encoding for CXCR4 and GFP genes, as previously described [
25]. For CXCR4 silencing, cells with exogenously expressed CXCR4 (RPMI8226-CXCR4) were stably transduced with lentiviral vectors encoding for specific anti-CXCR4 short hairpin RNA (shRNA) (pLKO.1-shRNA-CXCR4 TRCN, Mission TRC Sigma) using the same envelope and packaging constructs.
Analysis of surface markers
Expression levels of CXCR4 were evaluated by immune staining with allophycocyanin (APC)-conjugated anti-CXCR4 monoclonal antibody (12G5 clone) (eBioscience, USA). In primary MM samples, counterstaining with anti-CD138 fluorescein (FITC)-conjugated antibody (IQ products, Netherlands) was performed. The cells were analyzed by Navios (Becton Coulter), using Kaluza software.
XTT viability assay
Cells were exposed in vitro to increasing concentrations of AMG9810 for 48 h, and viability was determined as described in Additional file
1.
Cell cycle analysis
Cells were exposed in vitro to increasing concentrations of AMG9810 and bortezomib for 48 h and analyzed by FACS as described in Additional file
1.
Assessment of apoptosis
Apoptosis was determined as described in Additional file
1.
Quantitative RT-PCR analysis
RNA isolation and qRT-PCR analysis were performed as described in Additional file
1.
Acridine Orange staining
To assess vesicle acidification, MM cells were exposed to AMG9810 (10 µM), bortezomib, capsaicin or their combinations for different time points and were loaded with 1 µg/ml acridine orange (AO) (Sigma), for 30 min in 37 °C, and analyzed by flow cytometry.
Mitochondrial ROS accumulation
Following treatment with indicated reagents, levels of mitochondrial ROS were assessed using MitoSOX Red in combination with Annexin V-CF647 using FlowCellect™ MitoStress Kit (Merck Millipore) according to the manufacturer’s instructions.
Mitochondrial mass detection
Mitochondrial mass in live cells treated with indicated reagents was monitored using the probe MitoSpy™ Red CMXRos (Biolegend) at final concentration of 100 nM, according to the manufacturer’s instructions, and analyzed by flow cytometry.
Assessment of mitochondrial membrane potential (ΔΨm)
Effect of AMG9810 and bortezomib treatment on Δ
Ψm was evaluated using DiOC6 (Sigma-Aldrich) staining as previously described [
24].
Cell migration assay
Migration of MM cell lines in response to various CXCL12 concentrations (5–500 ng/ml) (PeproTech EC) was evaluated using 5-µm pore size transwells (Costar). The quantity of cells migrating within four hours to the lower compartment was determined by FACS and expressed as a percentage of the input. For TRPV1 inhibition, cells were pre-treated (30 min in 37 °C) with AMG9810 (10 µM) and subjected to migration.
Cell adhesion assay
Cell adhesion was determined as described in Additional file
1.
Assessment of lysosomal membrane permeabilization
MM cells were exposed to AMG9810 (5–10 µM), bortezomib (3–5 nM) or their combination for 24 or 48 h, labeled with LysoTracker (Cell Signaling Technology) for 30 min, 37 °C for 30 min, and analyzed by flow cytometry.
Immunofluorescent staining and microscopy
MM cells were exposed to AMG9810 (10 µM), in the absence or presence of BAPTA-AM (5 µM) for 1 h. Next the cells were seeded on poly-D-lysine pre-coated slides for 30 min and loaded with Rhod-2 calcium marker (Invitrogen) for additional 30 min. Next, the cells were fixed with 100% ice-cold methanol for 5 min, washed with PBSx1 and permeabilized with 0.5% saponin for 30 min. After blocking non-specific binding with 1% BSA for 1 h, anti-COX IV antibody (1:500) (Cell Signaling Technology) in 0.5% saponin-containing buffer was applied for 2-h incubation. Thereafter, the slides were washed with PBSx1 and incubated with secondary anti-rabbit (1:500), FITC-conjugated antibody for 1 h and subsequently counterstained with DAPI-containing mounting solution (Vector Laboratories). Stained cells and negative controls were evaluated using an Olympus BX53 microscope connected to an Olympus DP73 camera (Olympus, Melville, NY, USA). Images were captured for analysis using cellSens imaging software (Olympus, Melville, NY, USA).
Mitochondrial calcium and total cellular calcium measurements by flow cytometry
The mitochondrial calcium indicator Rhod-2, AM (Invitrogen), was used to assess mitochondrial calcium in MM cells. To evaluate total intracellular calcium levels, eFluor 514 (eBioscience™) calcium sensor dye was utilized. Cells were pre-treated with indicated treatments and then loaded with 10 μM Rhod-2 or 5 μM eFluor 514 for 30 min, washed with PBSx1 and analyzed by Navios (Beckman Coulter), using Kaluza software.
Immunoblot analysis
Mitochondria/cytosol fractionation was performed using commercial kit (Biovision) according to the manufacturer’s instructions. Total protein lysates (50–70 μg) or mitochondria/cytosol fractions (30 μg) were resolved by electrophoresis in 10% SDS-PAGE and transferred onto PVDF membranes. Blots were subjected to a standard immunodetection procedure using specific antibodies and the ECL substrate (Biological Industries). Signal was detected using a Bio-Rad image analyzer (Bio-Rad). The primary antibodies used were: CHOP, BCL-2, MCL-1, BCL-XL, phospho-Erk1/2, phospho-AKT, phospho-pS6, HSP70, HSP40, COX IV, AIF, PINK1, VDAC, LAMP1, ubiquitin, α-tubulin (Cell Signaling Technology), MCL-1 (Santa-Cruz) and β-actin (Sigma-Aldrich).
Mass spectrometry-based proteomics
RPMI8226 cells were treated with bortezomib (10 nM), AMG9810 (10 µM) or combination of both drugs and subjected to proteomic analysis (Smoler proteomics center, Technion). Briefly, the samples were digested by trypsin and analyzed by LC–MS/MS on Q Exactive plus (Thermo). The data were analyzed with MaxQuant 1.6.0.16 vs the Human Uniprot database. The identifications are filtered for proteins identified with false discovery rate (FDR) < 0.01 with at least 2 peptides in the project.
Murine xenograft models of disseminated human MM and drug treatment
NSG mice were maintained under defined flora conditions at the Hebrew University Pathogen-Free Animal Facility (Jerusalem, Israel). All experiments were approved by the Animal Care Committee of the Hebrew University. Mice were injected intravenously with RPMI8226-CXCR4 human cells (5 × 106/mouse). Endpoints were paraplegia and weight loss > 10%. The mice were killed on the same day the endpoint was reached. Disease was verified by measurement of human immunoglobulin in plasma of inoculated mice using the ELISA kit (Immunology Consultants Laboratory). To investigate the therapeutic potential of AMG9810 as a single agent or in combination with bortezomib, three days after inoculation with RPMI8226-CXCR4 cells, mice were randomized and treated with intraperitoneal (i.p.) injections of either AMG9810 (10 mg/kg) twice per week, subcutaneous injections of bortezomib (0.5 mg/kg) twice per week, or with a combination of both agents, for a total of 6 injections. Animals were killed 24 days after tumor inoculation.
Statistical analyses
Data are expressed as the mean ± standard deviation (SD) or standard error (SE). Statistical comparisons of means were performed by a two-tailed unpaired Student's t test or the Mann–Whitney U test.
Discussion
Despite the recent advances and improving the clinical outcomes of patients with myeloma, anti-MM treatment remains challenging and novel treatment strategies are urgently needed. Drug resistance remains a major challenge for MM cure.
Calcium is a critical secondary messenger that mediates various cellular processes in normal and cancer cells, keeping the balance between proliferation, differentiation and cell death [
40‐
42]. Calcium is also one of the critical regulators of cell migration of various cell types, including tumor cells [
40]. Furthermore, calcium signaling was proposed to play a role in drug resistance of cancer cells [
43]. TRPV1 has been recognized as an important regulator of intracellular calcium levels and was shown to be functionally expressed in various cancer cells. These findings prompted us to consider the TRPV1 pathway as a therapeutic target in MM.
Our data highlight the novel role of TRPV1-dependent signaling in MM cell growth and drug sensitivity. TRPV1 inhibition using pharmacological inhibitor AMG9810 resulted in calcium-dependent accumulation of mitochondrial ROS, followed by mitochondrial destabilization and MM cell death. These results are in accordance with previous findings showing that calcium acts as a key regulator of mitochondrial function and that its overload can impair electron transport leading to ROS generation [
26]. Furthermore, changes in mitochondrial calcium have been shown to regulate many cellular processes such as apoptosis [
44], autophagy [
45] and organelle crosstalk between mitochondria and ER [
46]. Excessive calcium in the mitochondria is known to be associated with sustained activation of mitochondrial membrane permeability and the release of apoptotic factors such as cytochrome c into the cytosol [
47]. Indeed, sustained activation of both apoptotic and autophagy pathways was observed upon TRPV1 inhibition in MM cells, including depletion of pro-apoptotic BCL-2 and MCL-1, increase in activated caspase 3 levels and increase in Annexin V staining. Additionally, increase in vesicle acidification and suppression of mTOR pathway suggested the activation of autophagy signaling following AMG9810 treatment. Furthermore, AMG9810-induced increase in CHOP indicated profound ER and mitochondrial stress mediated by TRPV1 inhibition.
Another important effect observed upon AMG9810 exposure was significant decrease in CXCR4 expression and function in MM cells, resulting in suppression of CXCL12-mediated signaling, while activation of TRPV1 using capsaicin enhanced CXCR4-mediated migration and adhesion. CXCR4/CXCL12 chemokine axis was shown to play pro-tumorigenic role in MM development and progression, inducing MM cell localization in the BM niche and promoting environment-mediated drug resistance [
48]. CXCR4 activity is known to be dependent on calcium flux [
49]. In normal and cancer cells, CXCL12 activation induces release of calcium from internal stores, triggering phospholipase C activation and the generation of inositol trisphosphate and diacylglycerol. Thus, calcium signaling is associated with processes that occur during metastasis, including cell migration and invasion [
50]. Indeed, previous work demonstrated that increased extracellular calcium levels and intracellular calcium flux upregulated CXCR4 expression and activity in CD34+ hematopoietic stem cells [
51]. In agreement with these findings, here we show that TRPV1 inhibition interferes with calcium signaling and suppresses both CXCR4 expression and activity in MM cells. On the contrary, TRPV1 activation using capsaicin promotes calcium influx, resulting in transient increase in cytosol calcium levels and, therefore, supporting CXCR4-mediated activity. Hence, our data demonstrate that TRPV1 is functionally connected to CXCR4-mediated activation, migration and adhesion of MM cells and reveal TRPV1 as a potential novel target in MM, possibly implicated in disease progression and refractoriness. Accordingly, inhibition of TRPV1 using AMG9810 effectively overcame stroma-mediated protection and restored the sensitivity of MM cells to bortezomib.
Moreover, combination of bortezomib with AMG9810 revealed synergistic anti-myeloma activity with unique mechanism. Simultaneous inhibition of proteasome and TRPV1 in MM cells promoted extensive mitochondrial damage with deleteriously increased mitochondrial ROS, induced lysosomal destabilization and uncompensated ER stress.
The major mechanism of bortezomib-induced cell death involves the accumulation of misfolded protein aggregates, which induce ER stress followed by an UPR. Activation of UPR may induce several lines of responses, acting to promote cellular survival or committing the cell to apoptosis. Increased expression of chaperons restores the folding capacity and ameliorate the stress, therefore reducing the responsiveness to bortezomib. Indeed, HSP70 induction is known to be a part of compensatory UPR in MM, while HSP70 inhibition was shown to be effective in combination with bortezomib [
52]. Furthermore, the increased levels of chaperones including HSP70 have been associated with resistance to proteasome inhibitors in MM cells [
53]. Our data show that TRPV1 inhibition using AMG9810 effectively downregulates HSP70 levels, overcoming the compensatory response induced by proteasome inhibitors bortezomib and carfilzomib. This may provide mechanistical hint, explaining the observed synergism between proteasome inhibitors and AMG9810 in MM cells. Noteworthy, the regulatory link between TRPV1 and HSP70 in epithelial cells was previously reported [
54].
Notably, in addition to cytosolic HSP70, profound increase in mitochondrial HSP70 levels was detected upon bortezomib exposure, suggesting the induction of mitochondrial UPR. The mtHSP70 machinery plays a critical role in maintaining proteostasis balance in the mitochondria and is induced upon proteotoxic stress [
55]. It was shown that elevated mtHSP70 prevents mitochondrial membrane depolarization and protects the cells from apoptosis by obliterating conformational changes in BAX and activating the AKT pathway [
56]. Overexpression of mtHSP70 in cancer cells causes resistance against chemotherapeutic drugs like cisplatin [
57], while reduction in mtHSP70 leads to increased apoptosis by decreasing mitochondrial membrane potential [
58]. However, the role of mtUPR in responses to proteasome inhibitor therapy remains mainly unexplored. Thus, our work provides the evidence for mtHSP70 increase and mtUPR induction upon bortezomib treatment and suggests its involvement in cell protection and drug resistance. Importantly, AMG9810 completely depleted bortezomib-induced mtHSP70 and therefore abrogated protective mtUPR.
Another important finding of our study indicates that bortezomib-induced mtUPR involves the accumulation of ubiquitin-protein conjugates in mitochondrial fraction following bortezomib exposure. Inhibition of the degradation of ubiquitin-labeled proteins is known consequence of proteasome inhibitors. Of note, AMG9810 in combination with bortezomib significantly downregulated the levels of proteins related to ubiquitination system, including ubiquitin ligases and ubiquitin activating enzymes. Accordingly, TRPV1 inhibition with AMG9810 significantly diminished the accumulation of poly-ubiquitinated proteins in both the cytosol and the mitochondria. Inhibition of the ubiquitination system was shown to be effective in combination with proteasome inhibitors. For example, lenalidomide, an inhibitor of E3 ubiquitin ligase cereblon, is potent anti-MM drug [
59]. Furthermore, ubiquitin-activating enzyme inhibition using TAK-243 molecule demonstrated anti-MM activity in preclinical models [
60]. Recent evidences show a regulatory role for calcium in modulating ubiquitination system. Thus, ubiquitination activity of numerous E3 ligases is regulated by calcium [
36]. Thereby, inhibition of ubiquitination by AMG9810 and potentiation of ER and mitochondrial stress by bortezomib can represent a mechanism that contribute to bortezomib/AMG9810 synergism. Furthermore, our data revealed increased basal level of protein ubiquitination in bortezomib-resistant cells, while AMG9810 in combination with bortezomib reduced the accumulation of ubiquitinated proteins in resistant cells. Therefore, modulation of the ubiquitination activity using TRPV1 inhibitor AMG9810 could be a new approach to target bortezomib-resistant MM cells.
Moreover, calcium is an important molecule of crosstalk at the ER-mitochondria junctions [
46]. Accordingly, it was shown that E3 ligase Mahogunin Ring Finger 1(MGRN1) regulates in a calcium-dependent manner the exchanges between the ER and mitochondria and influences mitochondrial quality control through mitophagy [
61]. Therefore, we hypothesize that TRPV1 inhibition results in sustained perturbation in intracellular calcium, interferes with ubiquitination activity and in combination with bortezomib promotes unresolved mitochondrial damage, inducing mitophagy in MM cells. This suggestion was further supported by our findings demonstrating the decrease in mitochondrial mass, mitochondrial-lysosomal fusion and mitochondrial accumulation of PINK1, a master regulator of UPR-induced mitophagy.
Finally, the effect of AMG9810 was validated in our in vivo model of CXCR4-driven human MM engrafting in murine BM. Our results demonstrate that single-agent treatment with AMG9810 targeted MM cells in the BM niche and significantly reduced tumor load. Most importantly, the combination of AMG9810 with bortezomib demonstrated preferential anti-MM activity, effectively reducing MM tumor load.
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