Exosome-Transmitted PSMA3 and PSMA3-AS1 Promote Proteasome Inhibitor Resistance in Multiple Myeloma

Author Manuscript Published OnlineFirst on January 4, 2019; DOI: 10.1158/1078-0432.
CCR-18-2363
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Exosome-Transmitted PSMA3 and PSMA3-AS1 Promotes
Proteasome Inhibitors Resistance in Multiple Myeloma

Hongxia Xu1,2, +, Huiying Han1, +, Sha Song1, Nengjun Yi3, Chen’ao Qian4, Yingchun
Qiu1, Wenqi Zhou1, Yating Hong5, Wenyue Zhuang6, Zhengyi Li7, Bingzong Li5,*,
Wenzhuo Zhuang1, *
1
Department of Cell Biology, School of Biology & Basic Medical Sciences, Soochow
University, Suzhou, China.
2
Xiangyang No.1 People's Hospital, Hubei University of Medicine, Xiangyang,
China.
3
Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL
35294, USA.
4
Department of Bioinformatics, School of Biology & Basic Medical Sciences,
Soochow University, Suzhou, China.
5
Department of Haematology, the Second Affiliated Hospital of Soochow University,
Suzhou, China.
6
Department of Molecular Biology, School of Laboratory Medicine of Beihua
University, Jilin, China.
7
Department of Clinical Examination Basis, Laboratory Acadamy of Jilin Medical
College, Jilin, China.
+
: Contribute equally to this work
*: Correspondence: Wenzhuo Zhuang, Email: zhuangwenzhuo@suda.edu.cn, Tel.:
86−512−65880103, Address: School of Biology & Basic Medical Sciences, Soochow
University, Ren Ai Road 199, Suzhou, 215123, China. Or Bingzong Li, Email:
lbzwz0907@hotmail.com, Tel.: 86−512−67784069, Address: The Second Affiliated
Hospital of Soochow University, San Xiang Road 1055, Suzhou, 215006, China.
Running title: Exosomal PSMA3 and PSMA3-AS1 in PIs resistance

Key words: Multiple myeloma, Proteasome inhibitors, Exosome, Long non-coding
RNA, Drug resistance
Financial support: This study was supported by Natural Science Foundation of

Jiangsu Province China (BK20161218, BK20161223), the Science and Technology
Development Project of Suzhou City (SS201856), National Natural Science
Foundation of China (81670191, 81673448), The Applied Basic Research Programs
of Suzhou City (SYS201546), Natural Science Foundation of Jilin Province
(20160101234JC).
Conflict of Interest statement: The authors declare no potential conflicts of interest.

Text word count: 4830, Number of figures: 6, Number of tables: 0.
1
Downloaded from clincancerres.aacrjournals.org on January 6, 2019. © 2019 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 4, 2019; DOI: 10.1158/1078-0432.CCR-18-2363
Translational Relevance
PIs resistance is a major challenge for MM. The bone marrow microenvironment
promotes myeloma cells survival and proliferation through the interaction between the
MSCs and MM cells. The bone marrow microenvironment promotes the interactions
between the MSCs and MM cells which permit MM to survive and proliferate. Here
we discovered that exosome-mediated transfer of PSMA3 (encodes proteasome
subunit α7) and lncPSMA3-AS1 from MSCs to MM cells contributed to PIs
resistance. PSMA3-AS1, which arises from the antisense strand of PSMA3, was
highly expressed in myeloma cells (r-MM) and MSCs (r-MSCs)-derived from
bortezomib-resistant patients. As a pair of protein-coding/non-coding antisense
transcripts, PSMA3 and PSMA3-AS1 were disordered concurrently and correlated
positively in MM cells, driving PIs sensitivity in MM. These results provided in vitro
and in vivo evidence that interference with exosome RNAs served as a promising
approach to overcome PIs resistance in MM. Moreover, our data indicated that
circulating exosomal PSMA3 and PSMA3-AS1 could develop the prognostic
stratification of MM patients, in addition to the international staging system.
Research.
Abstract
Purpose: How exosomal RNAs released within the bone marrow microenvironment
affect proteasome inhibitors (PIs) sensitivity of multiple myeloma (MM) is currently
unknown. This study aims to evaluate which exosomal RNAs are involved and by
which molecular mechanisms they exert this function.
Experimental Design: Exosomes were characterized by dynamic light scattering,
transmission electron microscopy and western blot. Coculture experiments were
performed to assess exosomal RNAs transferring from mesenchymal stem cells
(MSCs) to MM cells. The role of PSMA3-AS1 in PIs sensitivity was further evaluated
in vivo. To determine the prognostic significance of circulating exosomal PSMA3 and
PSMA3-AS1, a cohort of newly diagnosed MM patients was enrolled to study. Cox
regression models and Kaplan-Meier curves were used to analyze progression free
survival (PFS) and overall survival (OS).
Results: We identified that PSMA3 and PSMA3-AS1 in MSCs could be packaged
into exosomes and transferred to myeloma cells, thus promoted PIs resistance.
PSMA3-AS1 could form an RNA duplex with pre-PSMA3, which transcriptionally
promoted PSMA3 expression by increasing its stability. In xenograft models,
intravenously administered siPSMA3-AS1, was found to be effective in increasing
carfilzomib sensitivity. Moreover, plasma circulating exosomal PSMA3 and
PSMA3-AS1 derived from MM patients were significantly associated with PFS and
OS.
Research.
Conclusion: This study suggested a unique role of exosomal PSMA3 and
PSMA3-AS1 in transmitting PIs resistance from MSCs to MM cells, through a novel
exosomal PSMA3-AS1/PSMA3 signaling pathway. Exosomal PSMA3 and
PSMA3-AS1 might act as promising therapeutic targets for PIs resistance and
prognostic predictors for clinical response.
Introduction
Multiple myeloma (MM) is a malignancy of the plasma cell characterized by
proliferation of plasma cell clones (1). The proteasome inhibitor bortezomib (BTZ)
has shown promise in the treatment of MM (2). However, its therapeutic activity was
severely impeded by BTZ resistance (3). Multifactorial mechanisms underlying PIs
resistance have been studied: genetic mutations, gene expression signatures, copy
number abnormalities and bone marrow microenvironment (4). Nevertheless, MM is a
multifaceted disease related with genetic, epigenetic and chromosomal alterations,
and the mechanisms underlying PIs resistance in MM remains elusive.
The proteasome is a 26S enzyme complex that consists of a 19S regulatory complex
and a core 20S catalytic complex. The 20S complex is composed of two rings of
seven α subunits and two rings of seven β subunits (5). PIs could slightly inhibit the
peptidyl glutamyl–like activity at the β1 subunit (PSMB6) and mainly target the
chymotrypsin-like activity of β5 subunit (PSMB5) (6, 7). The mutations in β5 subunit
or the increased expression of β5 subunit was detected in BTZ-resistant hematologic
tumor cell lines (8). However, the β5 mutations and overexpression are absent in
clinical samples derived from BTZ resistant patients (9, 10). Therefore, the β5 subunit
Research.
dysregulation may not be uniquely responsible for PIs resistance. It is conceivable
that other mechanisms could be associated with PIs resistance.
The bone marrow microenvironment activates many pathways leading to disease
progression. MSCs support tumor cell growth, metastasis and evasion of the immune
system (11). The interaction between MSCs and myeloma cells performs a critical
role in MM pathogenesis, progress and chemotherapy resistance (12, 13). These
findings indicate a need for developing novel drugs to counteract these cancer–stroma
interactions.
Exosomes act as key communicators between tumor microenvironment and cancer
cells (14, 15). The interaction between MSCs and MM cells plays a crucial role in
MM pathogenesis and drug resistance by exosomes (16, 17). Recently, lncRNAs have
been reported to exist in exosomes (18, 19). LncRNAs are transcribed from thousands
of loci in mammalian genomes and function in a wide range of biological processes
(20, 21). In cancers, lncRNAs are emerging important regulators in oncogenic
pathways (22, 23). However, the role of lncRNAs in the MM pathogenesis and
progression has not been fully elucidated. In particular, how exosomal lncRNAs
derived from the bone marrow microenvironment contribute to PIs resistance is
unexplored in MM.
Abundant bidirectional transcription of eukaryotic genes has been explored across
genomes (24). Emerging evidence suggests that antisense lncRNAs play vital roles in
regulating their sense transcripts partners, in particular, affecting the expression of
their associated protein coding genes (24, 25). However, we know little about the
Research.
precise function of natural antisense transcripts and the molecular mechanisms of
their biogenesis in MM.
This study explored how the MSCs-derived exosomal lncRNAs conferred PIs
resistance to MM cells and through which molecular mechanisms they elicited this
function. Our data underscored that exosomal lncPSMA3-AS1 was the key
determinant in mediating resistance to PIs by regulating the stability of PSMA3,
which encodes proteasome subunit α7. Upregulation of PSMA3 led to the α-subunit–
related increase in chymotrypsin-like proteasome activity. These combined
approaches targeting PMSA3 and PSMA3-AS1 in conjunction with PIs could enhance
therapeutic efficacy. Likewise, our findings suggest that the circulating exosomal
PSMA3 and PSMA3-AS1 from the plasma of MM patients could improve to identify
the newly diagnosed patients with poor outcomes.

Materials and methods
Patient Samples
Samples were taken from newly diagnosed MM patients who were enrolled to study.
The study was conducted in accordance with the Declaration of Helsinki. Informed
written consent was obtained from each subject or each subject's guardian. The human
investigations were performed after approval by an institutional review board and in
accordance with an assurance filed with. The detailed clinical features of these
patients were listed in Table S1. Progression-free survival (PFS) was defined as the
time between initiation of bortezomib therapy and the date of first evidence of
progressive. Patients who were progression-free at the time of analysis were censored
Research.
using the time between initial treatment and last follow-up. More details of patient
samples are available in supplementary data.
Plasmids construction
Full-length cDNA (PMSA3 or PSMA3-AS1) was synthesized and subcloned into the
EcoRI and BamHI sites of Lenti-CMV-puro vector, the constructs were verified by
DNA sequencing.
Exosomes purification
Exosomes secreted by cultured cell lines and MSCs were isolated using ExoQuick™
solution (System Biosciences). Exosomes in the plasma were isolated using
ExoQuick-LP™ solution (System Biosciences). The detailed procedures of exosome
isolation are available in supplementary data. The exosome pellet was isolated and the
protein content of the exosome suspension was analyzed by BCA protein assay kit
(Beyotime Biotechnology).
Dynamic Light Scattering analysis (DLS)
Dynamic Light Scattering analysis was performed to characterize and quantify the
particle size distribution of exosomes (Zetasizer Nano S90).
In vivo xenograft studies
The animal experiments were performed after acquired permission from the local
Ethical Committee according to the institutional guidelines for the use of laboratory
animals. More details of in vivo xenograft studies are available in supplementary data.
Statistical analysis
Survival analysis was performed to investigate the associations between the censored
Research.
outcomes and the clinical variables and the expression measures of PSMA3 and
PSMA3-AS1 using the R package survival. PFS and OS were the primary outcomes
of interest. The clinical stage (ISS) was treated as a three-level factor and thus coded
as two dummy variables (ISS2 and ISS3) to compare Stage I with Stages II and III.
All other clinical variables and the expression measures of PSMA3 and PSMA3-AS1
were numeric and were standardized to facilitate the comparison of hazards ratios
(HR).
We first used univariate Cox survival regression to separately analyze the predictor
variables. To illustrate the impact of the PSMA3 and PSMA3-AS1, we partitioned the
patients to two groups based on the medians of the PSMA3 and PSMA3-AS1
expressions, and estimated the survival curves of these two groups of patients using
the Kaplan-Meier method and compared their difference using the log-rank test. We
then used multivariate Cox survival regression to jointly fit all the clinical variables
with either PSMA3 or PSMA3-AS1, which adjusted for the potential confounding
effects of the clinical variables on PSMA3 or PSMA3-AS1. By fitting these Cox
models, we obtained the estimates of hazards ratios (HR), their 95% confidence
intervals (CIs), the corresponding p-values, and Harrell’s concordance (C-index). We
also validated the prognostic values of these models using the leave-one-out
cross-validation.
Pearson correlation analysis was employed to determine the correlation between the
expressions of PSMA3 or PSMA3-AS1. Student’s t test was performed to analyze the
difference of mean values between two groups for other continuous outcomes.
Research.
Other detailed assays are available in supplementary data.
RESULTS
MSCs–derived exosomes were transferred to myeloma cells and conferred
proteasome inhibitors resistance to myeloma cells.
We first examined whether MSCs secreted exosomes. Primary r-MSCs obtained from
bortezomib-resistant patients, or s-MSCs obtained from bortezomib-sensitive patients
were cultured in serum-free medium for 48 hours. Then we isolated exosomes using
standard methods and confirmed their identity by transmission electron microscope
(Figure 1A). DLS analysis was performed to quantify the exosome size distribution
(Figure 1B). Western blot was used to confirm the expression of Flotillin-1 and
HSP70, which were generally used as exosome markers (Figure 1C).
Next we investigated whether MSCs–derived exosomes could be transmitted into
myeloma cells. Exosomes derived from MSCs were fluorescently labeled with
PKH67, and then cultured with MM.1S or U266. The ability of myeloma cells to take
MSCs–derived exosomes was confirmed using confocal microscope (Figure 1D,
Figure S1A) and flow cytometric assays (Figure 1E, Figure S1B). As the recipient
cells, myeloma cells showed the same uptake efficiency of exosomes from r-MSCs
and those from s-MSCs (Figure 1F). These results suggested that exosomes derived
from MSCs could be transmitted to myeloma cells, indicating a promising role in
regulating biologic functions of myeloma cells.
To further investigate the effect of MSCs-derived exosomes on the proteasome
Research.
inhibitors resistance, U266 or MM.1S cells were treated with or without different
amounts of exosomes in the presence of bortezomib or carfilzomib. R-MSCs-derived
exosomes significantly reduced proteasome inhibitors sensitivity (Figure 1G, Figure
S1C). However, no difference in the anti-proliferative activities of proteasome
inhibitors was observed between treatment with or without different amounts of
exosomes obtained from s-MSCs. These data demonstrated that exosomes derived
from r-MSCs, but not from s-MSCs reduced the proteasome inhibitors sensitivity in
MM cells.
Identifying mRNAs involved in bortezomib-resistant patients.
To explore the possible mechanisms of proteasome inhibitors resistance in multiple
myeloma, gene expression profiling of bortezomib-resistant patients (NC/PD) and
bortezomib-sensitive patients (CR/PR/MR) were analyzed using GEO database
(GSE9782). GSEA showed that the positive regulation of proteasome gene signatures
(the gene sets: BIOCARTA_PROTEASOME_PATHWAY,
WONG_PROTEASOME_GENE_MODULE, KEGG_PROTEASOME) were
significantly enriched in bortezomib-resistant samples (Figure 2A). A volcano plot
showed that three transcripts (PSMA3: fold change = 1.29/ p=0.0014, PSMA3-AS1:
fold change = 1.27/ p=0.000595, USP13: fold change = 1.257/ p=0.0013) were
significantly upregulated abundances in bortezomib-resistant samples compared to
bortezomib-sensitive samples (Figure 2B). Subsequently, we identified that PSMA3
and PSMA3-AS1, not USP13, were present in MSCs-derived exosomes (Figure 2C).
10
Research.
Then we focused on PSMA3 and PSMA3-AS1 transcripts. PSMA3 and PSMA3-AS1
expression were higher in bortezomib-resistant samples compared with
bortezomib-sensitive samples (Figure 2D). In addition, Kaplan-Meier analysis showed
that high PSMA3 expression in CD138+ MM cells was associated with decreased PFS
(p = 0.0307) and poor OS (p = 0.0328) (Figure 2E). Cox proportional hazards
regression analysis further confirmed that high PSMA3 could act as an independent
prognostic factor for MM patients with bortezomib therapy in a multivariate analysis
(p = 0.0013, HR = 1.3104, 95%CI = 1.1113-1.545).
To investigate whether the PSMA3 content of MM was involved in facilitating
multiple myeloma progression, we analyzed the datasets obtained from the Oncomine
database to determine the PSMA3 expression alterations in CD138+ cells in
monoclonal gammopathy of undetermined significance (MGUS), smoldering
myeloma (SM), multiple myeloma (MM) and plasma cell leukemia (PCL). As shown
in Figure 2F, PSMA3 levels were increased in CD138+ cells from SM compared to
those from MGUS (26), and the PSMA3 expressions were upregulated in CD138+
cells from MM compared to those from MGUS (Figure 2G) (27). Moreover, the
PSMA3 levels appeared a progressive increase in MGUS, MM and PCL (Figure 2H)
(28). These analyses consistently suggested that the expression levels of PSMA3 in
CD138+ cells were correlated with MM progression.
Consistent with this previously published study, our clinical data showed that the
mRNA levels of PSMA3 and PSMA3-AS1 were upregulated in CD138+ MM cells
derived from bortezomib resistant patients relative to those from bortezomib sensitive
11
Research.
patients (Figure 2I). To further explore the mechanisms of proteasome inhibitors
resistance, MM cell lines with a 4-fold or greater resistance to bortezomib,
carfilzomib or ixazomib were developed. The proteasome inhibitors resistant
(bortezomib resistance, BR; carfilzomib resistance, CR; ixazomib resistance, IR)
models consistently displayed upregulation of PSMA3 and PSMA3-AS1 transcript
levels (Figure 2J, Figure S2). In addition, r-MSCs had increased expression of
PSMA3 and PSMA3-AS1 compared to s-MSCs (Figure 2K). Moreover, the
expressions of PSMA3 and PSMA3-AS1 in MSCs were positively correlated with
those in CD138+ myeloma cells (Figure 2L). These results suggested that high levels
of PSMA3 and PSMA3-AS1 were associated with proteasome inhibitors resistance of
myeloma cells.
Exosome-mediated transfer of PSMA3 and lncPSMA3-AS1 from MSCs to MM
cells contributed to proteasome inhibitors resistance
Next we investigated whether exosome-transferred PSMA3 and PSMA3-AS1 could
confer proteasome inhibitors resistance to MM cells. We found that PSMA3 and
PSMA3-AS1 levels statistically increased in MM cells incubated with
r-MSCs-derived exosomes. This change did not occur when MM cells were incubated
with s-MSCs-derived exosomes (Figure 3A, Figure S3A). Moreover, U266 or MM.1S
incubated with exosomes from r-MSCs but not those from s-MSCs showed an
increase in the chymotrypsin-like (ChT-L) activity of the proteasome (Figure S3B).
Treatment directly with r-MSCs-derived exosomes or s-MSCs-derived exosomes had
12
Research.
no effects on cell viability and apoptosis (Figure S3C, S3D).
To further evaluate the possibility that exosome-mediated transmission of PSMA3 and
PSMA3-AS1 from MSCs to MM cells contributed to proteasome inhibitors resistance,
we examined whether the effect could be reversed when PSMA3 or PSMA3-AS1
expression was downregulated in r-MSCs. As expected, the levels of PSMA3 or
PSMA3-AS1 were decreased in MM cells upon incubation with exosomes from
PSMA3 or PSMA3-AS1-knockdown r-MSCs compared to siRNA control cells
(Figure 3B, Figure S3E). Moreover, U266 or MM.1S incubated with exosomes from
r-MSCs displayed decreased proteasome inhibitors sensitivity, which could be
abolished by downregulating PSMA3 or PSMA3-AS1 in r-MSCs (Figure 3C, Figure
S3F). Consistent with the loss of function experiments, when PSMA3 or PSMA3-AS1
was upregulated in s-MSCs, levels of PSMA3 or PSMA3-AS1 were increased in MM
cells incubated with exosomes from PSMA3 or PSMA3-AS1-overexpression s-MSCs
compared to vector control cells (Figure 3B, Figure S3E). In concordance with
upregulation of PSMA3 and PSMA3-AS1, cells showed decreased proteasome
inhibitors sensitivity (Figure 3C, Figure S3F). Similar results were obtained when
directly knocking down PSMA3 or PSMA3-AS1 in U266 or MM.1S (Figure 3D,
Figure S3G). These data suggested that PSMA3 or PSMA3-AS1 level differences in
MSCs derived-exosome content could mediate PIs resistance in MM cells.
Previous study has showed that PSMA3 which encoding the constitutive proteasome
component, α7 subunit, performed an important role in proteasome formation and
function (29). To further determine the role of PSMA3 and PSMA3-AS1 in
13
Research.
proteasome inhibitors sensitivity in MM cells, we examine whether PSMA3 and
PSMA3-AS1 levels were associated with proteasome activity. As expected,
knockdown of PSMA3 or PSMA3-AS1 caused a decrease in the chymotrypsin-like
(ChT-L) activity of the proteasome in U266 or MM.1S, whereas upregulated either
PSMA3 or PSMA3-AS1 enhanced the ChT-L proteasome activity (Figure 3E, Figure
S3H). These findings demonstrated that knockdown of either PSMA3 or PSMA3-AS1
increased the proteasome inhibitors sensitivity. Consistent with this finding,
overexpressing either PSMA3 or PSMA3-AS1 reduced the proteasome inhibitors
sensitivity. Taken together, these data indicated that the MSCs-derived exosomal
PSMA3 and PSMA3-AS1 conferred proteasome inhibitors resistance to the MM cells.
Higher levels of PSMA3 and PSMA3-AS1 were less sensitive to the proteasome
inhibitors, which produced a greater increase in cell proliferation and
chymotrypsin-like proteasome activity.
Next, we determined whether downregulation of PSMA3 and PSMA3-AS1 would
lead to restoration of PIs sensitivity in PIs resistance myeloma cells. Either
PSMA3-siRNA–dependent prevention or PSMA3-AS1-siRNA–dependent prevention
after PIs exposure was accompanied by both a significantly lower cell proliferation
(Figure 3G), higher fraction of apoptotic cells (Figure 3H) as well as decreased ChT-L
proteasome activity (Figure 3F). Collectively, these results further underscored the
involvement of PSMA3 and PSMA3-AS1 overexpression in conferring PIs resistance
and provided a targeted strategy to reverse PIs resistance.
14
Research.
PSMA3-AS1 reduced proteasome inhibitors sensitivity by regulating PSMA3
expression in MM cells.
In order to identify lncRNA PSMA3-AS1 involved in PIs resistance in MM, we
characterized the PSMA3-AS1. RNA fluorescence in situ hybridization (FISH) and
nuclear and cytoplasmic fractions assays indicated that PSMA3-AS1 existed both in
the nucleus and in the cytoplasm (Figure 4A, 4B). Rapid amplification of cDNA ends
(RACE) was performed for directional sequencing of 5′ and 3′ ends and two splice
variants, 2531 nt and 2440 nt were identified (Figure 4C). We found the presence of a
poly-A tail, indicating that PSMA3-AS1 is a fully processed transcript of RNA
polymerase II. Coding Potential Calculator (CPC, http://cpc.cbi.pku.edu.cn/) (30) and
Coding Potential Assessment Tool (CPAT, http://lilab.research.bcm.edu/cpat/) (31)
analyses displayed that PSMA3-AS1 has no coding potentiality (Table S4, Table S5).
Northern blotting assay further confirmed the existence of full length PSMA3-AS1 in
MM.1S (Figure 4D).
It is reported that the antisense transcripts can commonly regulate the sense transcripts
expressions in two ways (32). In concordant way, the antisense transcripts have
positive effects on sense transcripts (33). Conversely, the level of the sense RNA, or
corresponding protein level is negatively regulated by the antisense transcripts in
discordant way (34). To explore the biology of PSMA3-AS1 and consider its potential
significance, the correlation between PSMA3-AS1 and PSMA3 was examined in MM
cells derived from 57 MM patients by Pearson correlation analysis. The results
showed that PSMA3-AS1was positively correlated with PSMA3 in MM cells (Figure
15
Research.
4E).
To examine whether the PSMA3-AS1 transcript regulated the PSMA3 expression, we
transiently transfected siRNA PSMA3-AS1 to U266 or MM.1S. We found that
knockdown PSMA3-AS1 significantly decreased PSMA3 level in U266 or MM.1S
(Figure 4F, Figure S4A).
Given that PSMA3-AS1 knockdown decreased the proteasome activity and
subsequently reduced the cell viability (Figure 3D, 3E, Figure S3G, S3F), we sought
to determine whether reconstituting the expression of PSMA3 in PSMA3-AS1
downregulated cells could reverse the altered biological activity. We found that
restoring the expression of PSMA3 in PSMA3-AS1 knockdown cells significantly
abrogated the reduced ChT-L activity and the increased proteasome inhibitors
sensitivity conferred by PSMA3-AS1 downregulation in MM cells (Figure 4G, 4H,
Figure S4B, S4C). These data demonstrated that PSMA3-AS1 had effects on
proteasome activity and proteasome inhibitors sensitivity by regulating PSMA3
expression in MM cells.
PSMA3-AS1 formed an RNA duplex with PSMA3-AS1 pre-mRNA and increased
its stability.
Antisense transcripts commonly interact with their sense transcripts in a
double-stranded RNA structures form, which may potentially regulates the transport,
splicing and stability of the sense transcripts (24). PSMA3 and PSMA3-AS1 genes
are overlap and both located at chromosome 14p23.1. They are transcribed in opposite
16
Research.
directions. PSMA3-AS1 overlaps the 2029 nucleotides within the 7th intron of
PSMA3 (Figure S5A). To verify the existence of RNA duplex structure between
PSMA3-AS1 and pre-PSMA3, RNase protection assay was performed on RNAs from
U266 and MM.1S. RNase A digested the single-stranded RNA and the remaining
double-stranded RNA was protected from degradation. According to the PCR assays
using indicated primers, the overlapping part of both transcripts existed, which
suggesting that PSMA3-AS1 and pre-PSMA3 indeed form a RNA duplex (Figure
S5B). RNA-RNA duplex could change the secondary or tertiary structure of RNA and
protect RNA from RNase degradation, thereby increase its stability (33, 35). To
investigate whether the duplex form between PSMA3-AS1 and pre-PSMA3 affected
the PSMA3 mRNA stability, we assessed the stability of PSMA3 transcripts by
blocking new RNA synthesis with α-amanitin (an RNA polymerase II inhibitor) and
measuring the loss of PSMA3, PSMA3-AS1, GAPDH and 18s RNA over a 24h
period (Figure S5C). The results showed that knockdown PSMA3-AS1 decreased
stability of PSMA3. Conversely, PSMA3-AS1 overexpression increased stability of
PSMA3 (Figure S5D). Together, these results demonstrated that PSMA3-AS1 formed
an RNA duplex with PSMA3-AS1 pre-mRNA, which led to enhance the stability of
PSMA3 by reducing its decay.
Targeting PSMA3-AS1 increased proteasome inhibitors sensitivity in vivo.
To evaluate the therapeutic potential of PSMA3-AS1 in MM in vivo, we established
bioluminescent MM models (U266-luc), which recapitulates the bone
17
Research.
pathophysiology and the clinical sequelae observed in myeloma patients (36) (Figure
5A). The mRNA levels of PSMA3-AS1 and PSMA3 in the mouse CD138+ cells were
performed by qPCR to confirm the injected siRNA efficiency in vivo (Figure 5C).
Bioluminescent imaging revealed that therapeutic PSMA3-AS1 siRNA efficiently
increased the sensitivity of U266 xenografts to carfilzomib treatment (Figure 5B).
Moreover, the combination treatment with PSMA3-AS1 siRNA and carfilzomib
significantly prolonged the overall survival compared to carfilzomib combination
with siRNA control or siPSMA3-AS1-treated alone (Figure 5D). No significant
weight loss or unexpected deaths was found during treatment. Collectively,
downregulation of PSMA3-AS1 in vivo significantly enhanced the sensitivity of MM
xenografts to carfilzomib. These data revealed that PSMA3-AS1 was essential for
proteasome inhibitors resistance in MM cells.
Prognostic role of circulating exosomal PSMA3 and PSMA3-AS1 in multiple
myeloma
Several cell types, including cancer cells secrete exosomes. Exosomes have been
reported to be isolated from the peripheral blood and act as powerful markers for
disease progression in cancer (37, 38). To determine the prognostic significance of
circulating exosomal PSMA3 and PSMA3-AS1 in MM, we isolated and characterized
exosomes from the plasma of MM patients. A cohort of 57 newly diagnosed MM
patients was uniformly treated and followed. QPCR assays were performed to analyze
circulating exosomal RNAs derived from plasma of MM patients.
18
Research.
First, we isolated plasma circulating exosomes from MM patients and confirmed them
by transmission electron microscope (Figure 6A) and dynamic light scattering assays
(Figure 6B). Similar numbers of exosomes were isolated from bortezomib resistant
patients and bortezomib sensitive patients (Figure 6C). By comparing PSMA3 and
PSMA3-AS1 levels in circulating exosomes and the corresponding exosome-depleted
plasma, we found that circulating PSMA3 and PSMA3-AS1 predominantly existed in
exosomes (Figure 6D). Moreover, expressions of PSMA3 and PSMA3-AS1 in
circulating exosomes were positively correlated with that in CD138+ myeloma cells
(Figure 6E).
The results from the univariate and multivariate Cox survival regressions were present
in Table S6. We can see that Exosmic PSMA3 and PSMA3-AS1 were significantly
associated with both PFS and OS in the univariate and multivariate analyses (Table S6,
Figure 6F, 6G). We compared the C-indices of the models fitting the ISS alone, ISS
with PSMA3, and ISS with PSMA3-AS1 to show the predictive value of the PSMA3
and PSMA3-AS1 expressions (Table S7). In order to validate the prognostic values of
PSMA3 and PSMA3-AS1, the leave-one-out cross-validation was performed (Table
S7). The results showed that both exosomal PSMA3 and PSMA3-AS1 were
significant predictive biomarkers on PFS and OS. These data indicated that the
exosmal PSMA3 or PSMA3-AS1 could be crucial in identifying worse prognosis in
newly diagnosed MM patients. Therefore, it is advisable to evaluate PSMA3 and
PSMA3-AS1 expression in MM to identify patients who might benefit from
bortezomib therapy before deciding on a course of treatment.
19
Research.
Discussion
Resistance to the PIs is a major clinical problem in MM and the mechanisms have not
been completely revealed. The bone marrow microenvironment could promote the
survival and drug resistance of myeloma cells. It has been proposed that MSCs
contribute to MM microenvironment, where they can promote or suppress MM
progression (11, 39). We aimed to find a way to interfere these networks to stop tumor
growth and drug resistance.
Many types of cells could release exosomes (70–120 nm) into the extracellular
environment (38, 40). Exosomes are involved in intercellular communication, and in
this study we investigated how the transfer of exosomal PMSA3 and PSMA3-AS1
from MSCs to MM cells affected proteasome inhibitors resistance (Figure 6H).
Proteasome inhibitors resistance is a vital impediment to the clinical treatment.
Numerous underlying mechanisms have been identified (41-43), but the primary
cause is still unknown. Initial studies in hematologic tumor cell line models
demonstrated that overexpression of the β5 proteasome subunit or mutations in the β5
subunit’s bortezomib binding pocket were implicated in bortezomib resistance (8, 44).
However, the β5 mutations and overexpression are absent in clinical specimen
obtained from BTZ resistant patients (9, 10). To further elucidate mechanisms of PIs
resistance, we retrieved a database containing gene expression profile of 169
myeloma cases with clinical response and disease prognosis (45). The analysis of this
dataset showed that the mRNA levels of PSMA3 and PSMA3-AS1 in CD138+ cells
20
Research.
are upregulated in bortezomib-resistant patients. Moreover, increased PSMA3 and
PSMA3-AS1 mRNA expression correlated with proteasome inhibitors resistance and
poor outcome for myeloma patients treated with bortezomib. Further analyses of
Oncomine data showed that the PSMA3 levels appeared a progressive increase in
MGUS, SM, MM and PCL (26, 28). Similarly, our PIs resistant models (U266BR,
U266CR, U266IR, MM.1SBR, MM.1SCR, MM.1SIR) consistently displayed
up-regulation of PSMA3 and PSMA3-AS1 expression. These results suggested that
the PIs insensitivity in MM was due to up-regulated PSMA3 and PSMA3-AS1.
LncRNAs are extensively involved in cellular processes, including cell cycle,
apoptosis and immunity (23, 46). LncRNAs fulfill their functions through multiple
mechanisms, such as scaffolding of nuclear or cytoplasmic complexes, regulation of
gene expression and pairing with DNA or RNAs. Recently, several protein-coding
mRNAs have been reported to have natural antisense transcript partners, most of
which are noncoding RNAs (20, 47). Natural antisense lncRNA transcripts could
serve as therapeutic targets through regulating the sense gene expression (25, 48). In
this study, we identified a natural antisense transcript, PSMA3-AS1, arises from
intergenic regions of the PSMA3 gene. Our data indicated that as a pair of sense
protein-coding / antisense non-coding transcripts, PSMA3-AS1 could form a duplex
structure with PSMA3 pre-mRNA, thereby increasing its stability by reducing its
decay. And by enhancing the stability of PSMA3 pre-mRNA, PSMA3-AS1
upregulated the PSMA3 expression. In addition, downregulation of PSMA3-AS1
decreased PSMA3 expression, and subsequently reducing the chymotrypsin-like
21
Research.
proteasome activity and increasing the PIs sensitivity in myeloma cells. Importantly,
therapeutic lncPSMA3-AS1 siRNA efficiently increased the sensitivity of U266
xenografts to carfilzomib treatment, suggesting the significance of lncPSMA3-AS1 in
MM. Collectively, our data showed that disturbance of an antisense RNA
PSMA3-AS1 could alter the expression of sense mRNA PSMA3, suggesting that
antisense transcript PSMA3-AS1 contributed to the regulation of PSMA3 in MM cells.
These results suggested that many lncRNAs might exert their functions by RNA-RNA
direct interactions and that widely studying these interactions may further explore
lncRNA function and mechanism.
Exosomal RNAs have been reported to contribute to some aspects of oncology,
including tumorigenesis, metastasis and chemoresistance (15, 38). Recent evidence
suggests that more than twenty percent of exosomal RNAs are lncRNAs (49). Our in
vitro data indicated that lncPSMA3-AS1 could be packaged into exosomes and
transmitted to myeloma cells, conferred PIs resistance to MM cells by increasing the
PSMA3 expression.
Circulating exosomal RNAs have been implicated to act as potential non-invasive
molecular predictors of prognosis and drug response (50). However, the discovery of
specific exosomal RNAs as disease-specific biomarkers depends on validated
methods for sample isolation and analyses. Here, we identified circulating exosomal
PSMA3 and PSMA3-AS1 as clinically relevant biomarkers using standardized
techniques for sample collection and exosomal RNAs measurement. Another
challenge is that RNA copy numbers are generally low in circulating exosomes, which
22
Research.
makes it difficult to obtain sufficient RNA for analyses (50). In this study, we did
observe that PSMA3 and PSMA3-AS1 were relatively enriched in exosomes derived
from the plasma of MM patients by qPCR. Collectively, we identified that circulating
exosomal PSMA3 and PSMA3-AS1 derived from the plasma of MM patients were
associated with poor outcomes in regards to PFS and OS. The levels of exosomal
PSMA3 and PSMA3-AS1 could serve as prognostic factors for MM in a multivariate
Cox regression analysis. Our study provided evidence of the association between
circulating exosomal lncRNAs and outcomes in patients with newly diagnosed MM.
Here, we revealed that the informative lncRNAs derived from MSCs exosomes
involved in the communication between the MSCs and myeloma cells, thus resulted
in PIs resistance in MM. Our findings confirmed that PIs resistance was conferred by
increased expression of PMSA3 and PSMA3-AS1. The data also suggested that low
expression of PSMA3 and PSMA3-AS1 in pre-therapy MM exhibited a significant
improvement in prognosis after receiving bortezomib, whereas patients with high
PSMA3 and PSMA3-AS1 expressions were correlated with a poor bortezomib
response.
References
1.Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri A, et al. Review of 1027
patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003;78: 21-33.
2.Hideshima T, Chauhan D, Richardson P, Anderson KC Identification and validation of novel
therapeutic targets for multiple myeloma. J Clin Oncol 2005;23: 6345-6350.
23
Research.
3.Ahn JS, Jung SH, Yang DH, Bae SY, Kim YK, Kim HJ, et al. Patterns of relapse or
progression after bortezomib-based salvage therapy in patients with relapsed/refractory
multiple myeloma. Clin Lymphoma Myeloma Leuk 2014;14: 389-394.
4.Nikesitch N, Ling SC Molecular mechanisms in multiple myeloma drug resistance. J Clin
Pathol 2016;69: 97-101.
5.Chen P, Hochstrasser M Autocatalytic subunit processing couples active site formation in
the 20S proteasome to completion of assembly. Cell 1996;86: 961-972.
6.Kisselev AF, Callard A, Goldberg AL Importance of the different proteolytic sites of the
proteasome and the efficacy of inhibitors varies with the protein substrate. The Journal of
biological chemistry 2006;281: 8582-8590.
7.Crawford LJ, Walker B, Ovaa H, Chauhan D, Anderson KC, Morris TC, et al. Comparative
selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132.
Cancer research 2006;66: 6379-6386.
8.Oerlemans R, Franke NE, Assaraf YG, Cloos J, van Zantwijk I, Berkers CR, et al. Molecular
basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and
overexpression of PSMB5 protein. Blood 2008;112: 2489-2499.
9.Franke NE, Niewerth D, Assaraf YG, van Meerloo J, Vojtekova K, van Zantwijk CH, et al.
Impaired bortezomib binding to mutant beta5 subunit of the proteasome is the underlying basis
for bortezomib resistance in leukemia cells. Leukemia 2012;26: 757-768.
10.Lichter DI, Danaee H, Pickard MD, Tayber O, Sintchak M, Shi H, et al. Sequence analysis
of beta-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma
treated with bortezomib or dexamethasone. Blood 2012;120: 4513-4516.
24
Research.
11.Kuehl WM, Bergsagel PL Molecular pathogenesis of multiple myeloma and its
premalignant precursor. The Journal of clinical investigation 2012;122: 3456-3463.
12.McDonald MM, Fairfield H, Falank C, Reagan MR Adipose, Bone, and Myeloma:
Contributions from the Microenvironment. Calcif Tissue Int 2017;100: 433-448.
13.Tassone P, Neri P, Carrasco DR, Burger R, Goldmacher VS, Fram R, et al. A clinically
relevant SCID-hu in vivo model of human multiple myeloma. Blood 2005;106: 713-716.
14.Hsu YL, Hung JY, Chang WA, Lin YS, Pan YC, Tsai PH, et al. Hypoxic lung
cancer-secreted exosomal miR-23a increased angiogenesis and vascular permeability by
targeting prolyl hydroxylase and tight junction protein ZO-1. Oncogene 2017.
15.Shao H, Chung J, Lee K, Balaj L, Min C, Carter BS, et al. Chip-based analysis of exosomal
mRNA mediating drug resistance in glioblastoma. Nature communications 2015;6: 6999.
16.Roccaro AM, Sacco A, Maiso P, Azab AK, Tai YT, Reagan M, et al. BM mesenchymal
stromal cell-derived exosomes facilitate multiple myeloma progression. The Journal of clinical
investigation 2013;123: 1542-1555.
17.Wang J, Hendrix A, Hernot S, Lemaire M, De Bruyne E, Van Valckenborgh E, et al. Bone
marrow stromal cell-derived exosomes as communicators in drug resistance in multiple
myeloma cells. Blood 2014;124: 555-566.
18.Qu L, Ding J, Chen C, Wu ZJ, Liu B, Gao Y, et al. Exosome-Transmitted lncARSR
Promotes Sunitinib Resistance in Renal Cancer by Acting as a Competing Endogenous RNA.
Cancer cell 2016;29: 653-668.
19.Li Q, Shao Y, Zhang X, Zheng T, Miao M, Qin L, et al. Plasma long noncoding RNA
protected by exosomes as a potential stable biomarker for gastric cancer. Tumour biology : the
25
Research.
journal of the International Society for Oncodevelopmental Biology and Medicine 2015;36:
2007-2012.
20.Wang P, Xue Y, Han Y, Lin L, Wu C, Xu S, et al. The STAT3-binding long noncoding RNA
lnc-DC controls human dendritic cell differentiation. Science 2014;344: 310-313.
21.Scarola M, Comisso E, Pascolo R, Chiaradia R, Marion RM, Schneider C, et al. Epigenetic
silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA. Nature
communications 2015;6: 7631.
22.Liu B, Sun L, Liu Q, Gong C, Yao Y, Lv X, et al. A cytoplasmic NF-kappaB interacting long
noncoding RNA blocks IkappaB phosphorylation and suppresses breast cancer metastasis.
Cancer cell 2015;27: 370-381.
23.Wang Y, He L, Du Y, Zhu P, Huang G, Luo J, et al. The long noncoding RNA lncTCF7
promotes self-renewal of human liver cancer stem cells through activation of Wnt signaling.
Cell Stem Cell 2015;16: 413-425.
24.Faghihi MA, Wahlestedt C Regulatory roles of natural antisense transcripts. Nat Rev Mol
Cell Biol 2009;10: 637-643.
25.Carrieri C, Cimatti L, Biagioli M, Beugnet A, Zucchelli S, Fedele S, et al. Long non-coding
antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature
2012;491: 454-457.
26.Zhan F, Barlogie B, Arzoumanian V, Huang Y, Williams DR, Hollmig K, et al.
Gene-expression signature of benign monoclonal gammopathy evident in multiple myeloma is
linked to good prognosis. Blood 2007;109: 1692-1700.
27.Zhan F, Hardin J, Kordsmeier B, Bumm K, Zheng M, Tian E, et al. Global gene expression
26
Research.
profiling of multiple myeloma, monoclonal gammopathy of undetermined significance, and
normal bone marrow plasma cells. Blood 2002;99: 1745-1757.
28.Agnelli L, Mosca L, Fabris S, Lionetti M, Andronache A, Kwee I, et al. A SNP microarray
and FISH-based procedure to detect allelic imbalances in multiple myeloma: an integrated
genomics approach reveals a wide gene dosage effect. Genes Chromosomes Cancer
2009;48: 603-614.
29.Brehm A, Liu Y, Sheikh A, Marrero B, Omoyinmi E, Zhou Q, et al. Additive loss-of-function
proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production.
The Journal of clinical investigation 2015;125: 4196-4211.
30.Kong L, Zhang Y, Ye ZQ, Liu XQ, Zhao SQ, Wei L, et al. CPC: assess the protein-coding
potential of transcripts using sequence features and support vector machine. Nucleic Acids
Res 2007;35: W345-349.
31.Wang L, Park HJ, Dasari S, Wang S, Kocher JP, Li W CPAT: Coding-Potential Assessment
Tool using an alignment-free logistic regression model. Nucleic Acids Res 2013;41: e74.
32.Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, Nakamura M, et al. Antisense
transcription in the mammalian transcriptome. Science 2005;309: 1564-1566.
33.Faghihi MA, Modarresi F, Khalil AM, Wood DE, Sahagan BG, Morgan TE, et al. Expression
of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward
regulation of beta-secretase. Nat Med 2008;14: 723-730.
34.Wahlestedt C Natural antisense and noncoding RNA transcripts as potential drug targets.
Drug Discov Today 2006;11: 503-508.
35.Su WY, Li JT, Cui Y, Hong J, Du W, Wang YC, et al. Bidirectional regulation between
27
Research.
WDR83 and its natural antisense transcript DHPS in gastric cancer. Cell Res 2012;22:
1374-1389.
36.Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Shringarpure R, Akiyama M, et al.
Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic
strategy for multiple myeloma, other hematologic malignancies, and solid tumors. Cancer cell
2004;5: 221-230.
37.Manier S, Liu CJ, Avet-Loiseau H, Park J, Shi J, Campigotto F, et al. Prognostic role of
circulating exosomal miRNAs in multiple myeloma. Blood 2017;129: 2429-2436.
38.Zhou W, Fong MY, Min Y, Somlo G, Liu L, Palomares MR, et al. Cancer-secreted miR-105
destroys vascular endothelial barriers to promote metastasis. Cancer cell 2014;25: 501-515.
39.Reagan MR, Ghobrial IM Multiple myeloma mesenchymal stem cells: characterization,
origin, and tumor-promoting effects. Clinical cancer research : an official journal of the
American Association for Cancer Research 2012;18: 342-349.
40.Boelens MC, Wu TJ, Nabet BY, Xu B, Qiu Y, Yoon T, et al. Exosome transfer from stromal
to breast cancer cells regulates therapy resistance pathways. Cell 2014;159: 499-513.
41.Tomlin FM, Gerling-Driessen UIM, Liu YC, Flynn RA, Vangala JR, Lentz CS, et al. Inhibition
of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor
Cytotoxicity. ACS Cent Sci 2017;3: 1143-1155.
42.Bianchi G, Oliva L, Cascio P, Pengo N, Fontana F, Cerruti F, et al. The proteasome load
versus capacity balance determines apoptotic sensitivity of multiple myeloma cells to
proteasome inhibition. Blood 2009;113: 3040-3049.
43.Shabaneh TB, Downey SL, Goddard AL, Screen M, Lucas MM, Eastman A, et al. Molecular
28
Research.
basis of differential sensitivity of myeloma cells to clinically relevant bolus treatment with
bortezomib. PloS one 2013;8: e56132.
44.Lu S, Yang J, Chen Z, Gong S, Zhou H, Xu X, et al. Different mutants of PSMB5 confer
varying bortezomib resistance in T lymphoblastic lymphoma/leukemia cells derived from the
Jurkat cell line. Exp Hematol 2009;37: 831-837.
45.Mulligan G, Mitsiades C, Bryant B, Zhan F, Chng WJ, Roels S, et al. Gene expression
profiling and correlation with outcome in clinical trials of the proteasome inhibitor bortezomib.
Blood 2007;109: 3177-3188.
46.Hudson WH, Pickard MR, de Vera IM, Kuiper EG, Mourtada-Maarabouni M, Conn GL, et al.
Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional
repression and direct cell fate. Nature communications 2014;5: 5395.
47.He Y, Vogelstein B, Velculescu VE, Papadopoulos N, Kinzler KW The antisense
transcriptomes of human cells. Science 2008;322: 1855-1857.
48.Saldana-Meyer R, Gonzalez-Buendia E, Guerrero G, Narendra V, Bonasio R,
Recillas-Targa F, et al. CTCF regulates the human p53 gene through direct interaction with its
natural antisense transcript, Wrap53. Genes & development 2014;28: 723-734.
49.Huang X, Yuan T, Tschannen M, Sun Z, Jacob H, Du M, et al. Characterization of human
plasma-derived exosomal RNAs by deep sequencing. BMC genomics 2013;14: 319.
50.Bardelli A, Pantel K Liquid Biopsies, What We Do Not Know (Yet). Cancer cell 2017;31:
172-179.
Figure legends
Figure 1. MSCs derived exosomes were transferred to myeloma cells and

29
Research.
conferred proteasome inhibitors resistance to myeloma cells. (A) Representative
TEM imaging of exosomes derived from r-MSCs and s-MSCs (Bar = 100 nm). (B)
Exosomes isolated from r-MSCs and s-MSCs were measured by Dynamic Light
Scattering. (C) Representative western blot of HSP70 and flotillin-1 proteins in
r-MSCs-derived exosomes and s-MSCs-derived exosomes. (D) Myeloma cells were
cultured in the presence of MSCs–derived PKH67-labeled exosomes for 24hours.
Exosome uptakes by myeloma cells were shown using a confocal microscope
(original magnification, ×400). Myeloma cells were stained using DAPI (nuclei) and
594 conjugated anti-actin antibody. Scale bars, 10 μm. (E) Flow cytometric analysis
of U266 after incubation with fluorescently labeled exosomes. FL1 fluorescence
indicates exosome uptake. (F) Flow cytometric analysis of myeloma cells after
incubation with fluorescently labeled exosomes from r-MSCs and s-MSCs for
indicated time. (G) U266 and MM.1S cells were planted in 96-well plates with or
without different amounts of exosomes (5 μg, 20 μg, 40 μg) and bortezomib or
carfilzomib for 72h. The cell viability was measured using CCK-8. Error bars
represent the mean ± SD of 3 independent experiments, **, P < 0.01.
Figure 2. Identifying mRNAs involved in bortezomib-resistant patients. (A)
Specific baseline gene expression signatures are associated with proteasome activity.
Representative GSEA plots illustrating: The enrichment of genes involved in positive
regulation of proteasome among the transcripts in bortezomib-resistant patients. FDR,
false discovery rate; NES, normalized enrichment score. (B) Volcano plot showing the
30
Research.
gene expressions changes in the proteasome activity gene sets between
bortezomib-resistant samples and bortezomib-sensitive samples. PSMA3,
PSMA3-AS1 and USP13 which had a significantly altered expression were
highlighted in red. Vertical and horizontal dashed lines indicated cutoff values (p
value of 0.05 and absolute logarithmic fold change of 1.25). (C) 1/Ct values from
qPCR analysis to determine the loading efficiency of transcripts. The mean 1/Ct and
standard deviation of the three independent experiments are shown. (D) Analysis of
PSMA3 and PSMA3-AS1 expression in bortezomib-resistant MM (NC/PD) and
bortezomib-sensitive MM (CR/PR/MR) using the GEO database (GSE9782). (E)
Kaplan-Meier analysis of PFS and OS in the high and low PSMA3 groups according
to the median PSMA3 level in CD138+ MM cells. (F) (G) (H) PSMA3 mRNA
expression in CD138+ cells from MGUS, SM, MM or PCL was analyzed by using
Oncomine database. Data were presented as Log2 median-centered intensity. MGUS:
monoclonal gammopathy of undetermined significance, SM: smoldering myeloma,
MM: multiple myeloma, PCL: plasma cell leukemia. (I) Analysis of PSMA3 and
PSMA3-AS1 expression in bortezomib-resistant MM (SC/PD) and
bortezomib-sensitive MM (sCR/CR/VGPR/PR/MR) using our clinical data. RR:
relapsed/refractory patients, ND: newly diagnosed patients. (J) PSMA3 and
PSMA3-AS1 expression were determined in U266 drug-naïve, bortezomib-resistant
(B20R), carfilzomib-resistant (C40R), ixazomib-resistan (I20R) cells using qPCR.
Proteasome inhibitor resistant cells had increased expression of PSMA3 and
PSMA3-AS. (K) Analysis of PSMA3 and PSMA3-AS1 expression in r-MSCs and
31
Research.
s-MSCs. (L) Pearson correlation analysis between PSMA3 and PSMA3-AS1 levels in
CD138+ myeloma cells and that in MSCs. Error bars represent the mean ± SD of 3
independent experiments, *, P < 0.05, **, P < 0.01 and ***, P < 0.001.
Figure 3. Exosome-mediated transfer of PSMA3 and PSMA3-AS1 from MSCs to
MM cells contributed to proteasome inhibitors resistance. (A) QPCR analysis of
PSMA3 and PSMA3-AS1 expression in U266 after incubation with indicated
exosomes. R-exo: R-MSCs-derived exosomes; S-exo: S-MSCs-derived exosomes. (B)
QPCR analysis of PSMA3 in U266 after incubation with indicated exosomes.
oePSMA3: PSMA3-overexpressing; oePSMA3-AS1: PSMA3-AS1-overexpressing;
oeVec: empty vector. (C) MSCs were transfected with indicated siRNAs or
overexpressing vectors for 24 hours, and exosomes were isolated. These exosomes
were then treated with RNase to remove unincorporated RNAs. Cell viability of U266
was assessed 48 hours after incubation with or without indicated exosomes and
bortezomib using CCK-8 assay. (D) U266 cells were transfected with PSMA3 siRNA,
PSMA3-AS1 siRNA, control siRNA, PSMA3-overexpressing vector,
PSMA3-AS1-overexpressing vector or empty vector and treated with or without
bortezomib. The cell viability was measured using CCK-8. (E) U266 cells were
transfected with PSMA3 siRNA, PSMA3-AS1 siRNA, control siRNA,
PSMA3-overexpressing vector, PSMA3-AS1-overexpressing vector or empty vector.
The cell numbers were counted and a proteasome activity assay was done. Fold
changes of activity against no-treatment control was calculated. (F) (G) (H) After
32
Research.
transfection of with PSMA3 siRNA, PSMA3-AS1 siRNA, control siRNA, PIs
resistance cells were incubated (24-48 hours) with bortezomib, carfilzomib or
ixazomib, respectively. The proteasome activity (F) and cell viability (G) were
measured as described previously. The apoptosis was measured by Annexin V
/7-AAD apoptosis detection kit (H). Error bars represent the mean ± SD of 3
independent experiments, *, P < 0.05, **, P < 0.01 and ***, P < 0.001.
Figure 4. PSMA3-AS1 reduced proteasome inhibitors sensitivity by regulating
PSMA3 expression in MM cells. Characterization of lncRNA PSMA3-AS1. The
nuclear and cytoplasmic location of PSMA3-AS1 in MSCs. (A) RNA FISH assay of
PSMA3-AS1 in U266, MM.1S and MSCs. Scale bars, 10 μm. (B) QPCR detection of
PSMA3-AS1 in the cytoplasmic (C) and nuclear (N) fractions. GAPDH served as an
internal normalization control. (C) Gel electrophoresis of nested PCR products from
5'-RACE and 3'-RACE. (D) Northern blot analysis with in vitro–transcribed
strand-specific RNA probes for PSMA3-AS1 in MM.1S. β-actin serves as a loading
control. (E) Correlation between PSMA3 and PSMA3-AS1 in CD138+ myeloma cells
from myeloma patients. r = 0.538, p < 0.001. (F) U266 or MM.1S were transiently
transfected with PSMA3-AS1siRNA or a control siRNA. QPCR was performed to
determine gene levels, with GAPDH used as an internal normalization control.
PSMA3 protein expression was determined by western blot. (G) U266 cells were
transfected with PSMA3-AS1 siRNA, control siRNA with or without
PSMA3-overexpressing vector or empty vector. The cell numbers were counted and
33
Research.
the proteasome activity assay were done. Fold changes of activity against
no-treatment control was calculated. (H) U266 cells were treated with or without
bortezomib and transfected with PSMA3-AS1 siRNA or control siRNA, with or
without PSMA3-overexpressing vector or empty vector. The cell viability was
measured using CCK-8. Error bars represent the mean ± SD of 3 independent
experiments, *, P < 0.05, **, P < 0.01 and ***, P < 0.001
Figure 5. Targeting PSMA3-AS1 increased proteasome inhibitors sensitivity in
vivo. (A) NSG mice were transplanted with U266-luc+cells and the combined
chemotherapy regimens were started on day 15 post xeno-transplantation as indicated
in the upper panel. (B) Representative whole-body bioluminescence images of NSG
mice. Therapy of carfilzomib in combination with PSMA3-AS1 siRNA or siRNA
control or PSMA3-AS1 siRNA alone in vivo was performed. Tumor development was
monitored by noninvasive bioluminescence imaging. (n = 5/group). Error bars
represent the mean ± SD of 3 independent experiments, *, P < 0.05, **, P < 0.01. (C)
QPCR was performed to determine gene levels in mouse CD138+ cells, with GAPDH
used as an internal normalization control. Error bars represent the mean ± SD of 3
independent experiments, **: p < 0.01, ***: p < 0.001 versus vehicle control. (D)
Survival curves (Kaplan-Meier) of mice showed prolongation of overall survival with
indicated treatments (log-rank test, p < 0.01).
Figure 6. Exosomal PSMA3 and PSMA3-AS1 levels in plasma and CD138+ MM
34
Research.
cells correlate with BTZ response in MM Patients. (A) (B) Characterization of
circulating exosomes from the plasma of human subjects. (A) Representative TEM
imaging of exosomes from the plasma of human subjects (Bar = 100 nm). (B)
Exosomes from the plasma of human subjects were measured by DLS. (C) The
number of exosomes isolated from bortezomib resistant patients and bortezomib
sensitive patients was determined. SP: bortezomib sensitive patients, RP: bortezomib
resistance patients. (D) Circulating PSMA3 and PSMA3-AS1 predominantly exists in
exosomes. Exosomes isolated from three cases of MM patient plasma as well as the
corresponding exosome depleted plasma were subjected to RNA extraction and
RT-qPCR. The ratio of mRNAs level in the exosomes to those in exosome-free
fraction was shown. Relative gene expression values in the same volume of plasma
were normalized to exogenous reference λ polyA. (E) Pearson correlation analysis
between PSMA3 and PSMA3-AS1 levels in CD138+ myeloma cells and that in
circulating exosomes of MM patients (n = 57). Relative gene expression values in the
same volume of plasma were normalized to exogenous reference λ polyA. Results are
presented as mean ± -AS1 on
overall progression-free survival (PFS) and survival (OS). (G) The prognostic impact
of exosomal PSMA3 on overall progression-free survival (PFS) and survival (OS). (H)
A schematic diagram depicting the mechanism of transferring of PSMA3 and
PSMA3-AS1 by exosome conferring PIs resistance to MSCs.
35
Research.
Research.
Research.
Research.
Research.
Research.
Research.
Exosome-Transmitted PSMA3 and PSMA3-AS1 Promotes

Proteasome Inhibitors Resistance in Multiple Myeloma
Hongxia Xu, Huiying Han, Sha Song, et al.
Clin Cancer Res Published OnlineFirst January 4, 2019.
Updated version Access the most recent version of this article at:
doi:10.1158/1078-0432.CCR-18-2363
Supplementary Access the most recent supplemental material at:

Material http://clincancerres.aacrjournals.org/content/suppl/2019/01/04/1078-0432.CCR-18-2363.DC1
Author Author manuscripts have been peer reviewed and accepted for publication but have not yet
Manuscript been edited.
E-mail alerts Sign up to receive free email-alerts related to this article or journal.
Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.
Permissions To request permission to re-use all or part of this article, use this link
http://clincancerres.aacrjournals.org/content/early/2019/01/04/1078-0432.CCR-18-2363.
Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
Rightslink site.
Research.

Exosome-Transmitted PSMA3 and PSMA3-AS1 Promote Proteasome Inhibitor Resistance in Multiple Myeloma

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Exosome-Transmitted PSMA3 and PSMA3-AS1 Promote Proteasome Inhibitor Resistance in Multiple Myeloma

Uploaded by

Copyright:

Available Formats

Author Manuscript Published OnlineFirst on January 4, 2019; DOI: 10.1158/1078-0432.

Exosome-Transmitted PSMA3 and PSMA3-AS1 Promotes

Proteasome Inhibitors Resistance in Multiple Myeloma

Running title: Exosomal PSMA3 and PSMA3-AS1 in PIs resistance

Financial support: This study was supported by Natural Science Foundation of

Conflict of Interest statement: The authors declare no potential conflicts of interest.

we discovered that exosome-mediated transfer of PSMA3 (encodes proteasome

subunit α7) and lncPSMA3-AS1 from MSCs to MM cells contributed to PIs

highly expressed in myeloma cells (r-MM) and MSCs (r-MSCs)-derived from

bortezomib-resistant patients. As a pair of protein-coding/non-coding antisense

transcripts, PSMA3 and PSMA3-AS1 were disordered concurrently and correlated

circulating exosomal PSMA3 and PSMA3-AS1 could develop the prognostic

stratification of MM patients, in addition to the international staging system.

affect proteasome inhibitors (PIs) sensitivity of multiple myeloma (MM) is currently

which molecular mechanisms they exert this function.

Experimental Design: Exosomes were characterized by dynamic light scattering,

transmission electron microscopy and western blot. Coculture experiments were

performed to assess exosomal RNAs transferring from mesenchymal stem cells

in vivo. To determine the prognostic significance of circulating exosomal PSMA3 and

PSMA3-AS1, a cohort of newly diagnosed MM patients was enrolled to study. Cox

survival (PFS) and overall survival (OS).

Results: We identified that PSMA3 and PSMA3-AS1 in MSCs could be packaged

PSMA3-AS1 could form an RNA duplex with pre-PSMA3, which transcriptionally

promoted PSMA3 expression by increasing its stability. In xenograft models,

intravenously administered siPSMA3-AS1, was found to be effective in increasing

carfilzomib sensitivity. Moreover, plasma circulating exosomal PSMA3 and

Conclusion: This study suggested a unique role of exosomal PSMA3 and

PSMA3-AS1 in transmitting PIs resistance from MSCs to MM cells, through a novel

exosomal PSMA3-AS1/PSMA3 signaling pathway. Exosomal PSMA3 and

prognostic predictors for clinical response.

Multiple myeloma (MM) is a malignancy of the plasma cell characterized by

severely impeded by BTZ resistance (3). Multifactorial mechanisms underlying PIs

number abnormalities and bone marrow microenvironment (4). Nevertheless, MM is a

multifaceted disease related with genetic, epigenetic and chromosomal alterations,

and the mechanisms underlying PIs resistance in MM remains elusive.

chymotrypsin-like activity of β5 subunit (PSMB5) (6, 7). The mutations in β5 subunit

or the increased expression of β5 subunit was detected in BTZ-resistant hematologic

dysregulation may not be uniquely responsible for PIs resistance. It is conceivable

that other mechanisms could be associated with PIs resistance.

The bone marrow microenvironment activates many pathways leading to disease

role in MM pathogenesis, progress and chemotherapy resistance (12, 13). These

Exosomes act as key communicators between tumor microenvironment and cancer

of loci in mammalian genomes and function in a wide range of biological processes

(20, 21). In cancers, lncRNAs are emerging important regulators in oncogenic

derived from the bone marrow microenvironment contribute to PIs resistance is

Abundant bidirectional transcription of eukaryotic genes has been explored across

regulating their sense transcripts partners, in particular, affecting the expression of

precise function of natural antisense transcripts and the molecular mechanisms of

their biogenesis in MM.

determinant in mediating resistance to PIs by regulating the stability of PSMA3,

related increase in chymotrypsin-like proteasome activity. These combined

the newly diagnosed patients with poor outcomes.

investigations were performed after approval by an institutional review board and in

samples are available in supplementary data.

solution (System Biosciences). Exosomes in the plasma were isolated using

ExoQuick-LP™ solution (System Biosciences). The detailed procedures of exosome

Dynamic Light Scattering analysis (DLS)

particle size distribution of exosomes (Zetasizer Nano S90).

In vivo xenograft studies

effects of the clinical variables on PSMA3 or PSMA3-AS1. By fitting these Cox

intervals (CIs), the corresponding p-values, and Harrell’s concordance (C-index). We

expressions of PSMA3 or PSMA3-AS1. Student’s t test was performed to analyze the

Other detailed assays are available in supplementary data.

MSCs–derived exosomes were transferred to myeloma cells and conferred