MYC Causes Multiple Myeloma Progression via Attenuating TP53-Induced MicroRNA-34 Expression

MicroRNAs (miRNAs and miRs) are small (19–25 base pairs) non-coding RNAs with the ability to modulate gene expression. Previously, we showed that the miR-34 family is downregulated in multiple myeloma (MM) as the cancer progressed. In this study, we aimed to clarify the mechanism of miRNA dysregulation in MM. We focused particularly on the interaction between MYC and the TP53-miR34 axis because there is a discrepancy between increased TP53 and decreased miR-34 expressions in MM. Using the nutlin-3 or Tet-on systems, we caused wild-type (WT) p53 protein accumulation in human MM cell lines (HMCLs) and observed upregulated miR-34 expression. Next, we found that treatment with an Myc inhibitor alone did not affect miR-34 expression levels, but when it was coupled with p53 accumulation, miR-34 expression increased. In contrast, forced MYC activation by the MYC-ER system reduced nutlin-3-induced miR-34 expression. We also observed that TP53 and MYC were negatively correlated with mature miR-34 expressions in the plasma cells of patients with MM. Our results suggest that MYC participates in the suppression of p53-dependent miRNA expressions. Because miRNA expression suppresses tumors, its inhibition leads to MM development and malignant transformation.


Introduction
Cancer pathogenesis and progression are largely driven by mutations that cause abnormal gene expression and functional alterations [1,2]. However, epigenetic changes that regulate gene expression, including DNA promoter methylation and histone modification, also play important roles in oncogenesis [2,3]. We now know that non-coding RNAs are influential in epigenetics; despite previously being considered as 'junk RNAs' because they are not translated into proteins, many of these RNAs actually regulate gene expressions [4]. In particular, microRNAs (miRNAs)-small non-coding RNAs of 19-25 nucleotides-silence gene expressions by degrading target messenger RNA (mRNA) or by inhibiting its translation [4,5].
Multiple myeloma (MM) is a plasma cell malignancy [6,7]. Almost all patients with MM progress from a pre-malignant stage called monoclonal gammopathy of undetermined significance (MGUS). Chromosomal abnormalities are involved in the genesis of clonal plasma cells, and oncogene mutations, such as RAS mutations, play critical roles in disease progression [8]. However, all the steps in the progression from MGUS to MM have not been fully elucidated.

Patients
The study used bone marrow plasma cells from 109 patients with newly diagnosed MM and from 64 patients with MGUS. Plasma cells were purified from bone marrow mononuclear cells using anti-CD138 antibody conjugated with phycoerythrin (PE) (Beckman-Coulter, Brea, CA, USA) and an Easy Sep PE positive selection kit containing anti-PE antibody conjugated with micro-magnetic beads (STEMCELL Technologies, Vancouver, BC, Canada). The patients were diagnosed with MM or MGUS between July 2010 and March 2015. This study was approved by the institutional review board of Gunma University Hospital and followed all guidelines under the Declaration of Helsinki. Informed consent was obtained from all patients. The patients' demographics are shown in Table 1.
The three cell lines were cultured with 1 µg/mL doxycycline (TaKaRa Bio, Kyoto, Japan). Gene expression after 24 h of treatment was determined using real-time PCR. Cell proliferation was determined using WST-8 assays.

Isolation of Nucleic Acids
Total RNA, including miRNA, was extracted from the myeloma cell lines using a mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). RNA quantity and quality were measured using BioSpec-nano (SHIMADZU, Kyoto, Japan). Complementary DNA (cDNA) was synthesized using a PrimeScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa Bio, Kyoto, Japan). MicroRNA cDNA was produced using a TaqMan TM MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Tokyo, Japan).

Apoptosis Analysis
The myeloma cell lines were collected after treatment with the agents, washed twice with cold-PBS, and washed once with 7-AAD binding buffer (10 mM HEPES pH7.4, 140 mM NaCl, 2.5 mM CaCl 2 ). After centrifugation and aspiration, 5 µL 7-AAD (BioLegend, San Diego, CA, USA) and/or 5 µL Annexin V conjugated to FITC (BioLegend, San Diego, CA, USA) was added to the suspension. The antibodies were incubated for 30 min at RT and then washed once in PBS. The suspension after centrifugation and aspiration was analyzed on a BD FACSCanto TM II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Statistical Analysis
All statistical analyses were performed in EZR version 1.54 (Saitama, Japan) [19]. Significance was set at p < 0.05. The RT-qPCR data were analyzed using Student's t test or the Mann-Whitney U test. Correlations were evaluated using Spearman's rank method.

miR-34 Family and TP53 mRNA Expressions and Their Correlations in the Patients
Mature miR-34a and 34b and TP53 mRNA expressions in the bone marrow plasma cells were determined using RQ-PCR. Consistent with our previous data, both mature miR-34a and 34b expressions were lower in the MM plasma cells than in the MGUS plasma cells (p = 0.0063 and p < 0.001, respectively) ( Figure 1A,B). However, TP53 mRNA expressions were higher in MM than in MGUS (p = 0.0028) ( Figure 1C). Contrary to the expectation, in the plasma cells obtained from the MM and MGUS bone marrow specimens, a negative correlation between TP53 and mature miR-34a and 34b was observed (miR-34a: r = −0.402, p < 0.001, miR-34b: r = −0.341, p < 0.001) ( Figure 1D,E). These results imply that miR-34 expression is not upregulated by TP53.
nificance was set at p < 0.05. The RT-qPCR data were analyzed using Student's t test or the Mann-Whitney U test. Correlations were evaluated using Spearman's rank method.

miR-34 Family and TP53 mRNA Expressions and Their Correlations in the Patients
Mature miR-34a and 34b and TP53 mRNA expressions in the bone marrow plasma cells were determined using RQ-PCR. Consistent with our previous data, both mature miR-34a and 34b expressions were lower in the MM plasma cells than in the MGUS plasma cells (p = 0.0063 and p < 0.001, respectively) ( Figure 1A,B). However, TP53 mRNA expressions were higher in MM than in MGUS (p = 0.0028) ( Figure 1C). Contrary to the expectation, in the plasma cells obtained from the MM and MGUS bone marrow specimens, a negative correlation between TP53 and mature miR-34a and 34b was observed (miR-34a: r = -0.402, p < 0.001, miR-34b: r = -0.341, p < 0.001) ( Figure 1D,E). These results imply that miR-34 expression is not upregulated by TP53.

MYC mRNA Expression in Patients
As described above, forced TP53 expression can induce miR-34 expression. Thus, the TP53-miR-34 pathway may be inhibited by factors that are highly expressed/highly active in the patient specimen. MYC is a known driver oncogene for MM progression and a re-

MYC mRNA Expression in Patients
As described above, forced TP53 expression can induce miR-34 expression. Thus, the TP53-miR-34 pathway may be inhibited by factors that are highly expressed/highly active in the patient specimen. MYC is a known driver oncogene for MM progression and a repressor of several microRNAs, such as the miR-29 family. So, we next examined MYC expressions in MM and MGUS. As expected, MYC expression was significantly higher in MM than in MGUS (p = 0.001) ( Figure 4A). A weak negative correlation between MYC and miR-34a and 34b was observed (miR-34a: r = −0.30, p < 0.001; miR-34b: r = −0.19, p = 0.015) ( Figure 4B,C).

Myc Inhibitor Alone Did Not Change miR-34 Family Expression in Most HMCLs
Because MYC suppresses miR-29 family expression, we attempted to clarify whether MYC is involved in regulating miR-34 family expression. Human MCLs were cultured with the Myc inhibitor 10058-F4, which attenuates MYC transcriptional function through dissociating the MYC-MAX transcription factor complex. The Myc inhibitor alone did not significantly increase pri-miR-34a or mature miR-34a in KMS27, KMS28BM, and KMS26, except for in OPM2 cells ( Figure 5A

Myc Inhibitor Alone Did Not Change miR-34 Family Expression in Most HMCLs
Because MYC suppresses miR-29 family expression, we attempted to clarify whether MYC is involved in regulating miR-34 family expression. Human MCLs were cultured with the Myc inhibitor 10058-F4, which attenuates MYC transcriptional function through dissociating the MYC-MAX transcription factor complex. The Myc inhibitor alone did not significantly increase pri-miR-34a or mature miR-34a in KMS27, KMS28BM, and KMS26, except for in OPM2 cells ( Figure 5A-E).

Myc Inhibitor Alone Did Not Change miR-34 Family Expression in Most HMCLs
Because MYC suppresses miR-29 family expression, we attempted to clarify whether MYC is involved in regulating miR-34 family expression. Human MCLs were cultured with the Myc inhibitor 10058-F4, which attenuates MYC transcriptional function through dissociating the MYC-MAX transcription factor complex. The Myc inhibitor alone did not significantly increase pri-miR-34a or mature miR-34a in KMS27, KMS28BM, and KMS26, except for in OPM2 cells ( Figure 5A-E).

WT p53 Accumulation and Myc Inhibition Synergistically Upregulated miR-34 Expression
Because p53 induces the miR-34 family, we investigated whether p53 accumulation and simultaneous Myc inhibition would have a synergistic effect on miR-34 expression.

WT p53 Accumulation and Myc Inhibition Synergistically Upregulated miR-34 Expression
Because p53 induces the miR-34 family, we investigated whether p53 accumulation and simultaneous Myc inhibition would have a synergistic effect on miR-34 expression.
In the KMS26 and OPM2 cells with the Tet-on TP53 system, Myc inhibition further increased pri-miR-34a expression due to forced p53 overexpression. Mature miR-34a expression also markedly increased in the KMS26 cells, indicating a synergistic effect between p53 accumulation and Myc inhibition ( Figure 7A,B). The treatment of the Tet-on p53 KMS26 and OPM2 cells with doxycycline and the Myc inhibitor began at the same time. and simultaneous Myc inhibition would have a synergistic effect on miR-34 expression.
In the MM.1S cells, Myc inhibition enhanced the upregulation of pri-miR-34a and mature miR-34a induced by p53 accumulation from nutlin-3 treatment ( Figure 6A). Myc inhibition also increased pri-miR-34a expressions in KMS27 and KMS28BM cells following nutlin-3 treatment ( Figure 6B,C). The treatment of the MM.1S, KMS27, and KMS28BM cells with (-)-Nutlin-3 and the Myc inhibitor 10058-F4 began at the same time. In the KMS26 and OPM2 cells with the Tet-on TP53 system, Myc inhibition further increased pri-miR-34a expression due to forced p53 overexpression. Mature miR-34a expression also markedly increased in the KMS26 cells, indicating a synergistic effect between p53 accumulation and Myc inhibition ( Figure 7A,B). The treatment of the Tet-on p53 KMS26 and OPM2 cells with doxycycline and the Myc inhibitor began at the same time.

Forced MYC Activation Repressed p53-Mediated miR-34 Expression in MYC-ER Cell Lines
Because Myc inhibition enhances p53-mediated miR-34 family expression, we examined whether MYC activation could suppress p53-induced miR-34 family expression. We used an MYC-ER cell line that expresses MYC but masks its nuclear translocation signal with the estrogen receptor ligand binding domain (ER). When tamoxifen (4OHT) binds to ER, the nuclear translocation signal is unmasked, allowing Myc translocation to the nucleus and increased activity [17,18]. We simultaneously induced p53 accumulation and MYC activation in three MYC-ER cell lines, namely, osteosarcoma cell line (1) U2OS harboring WT-TP53, and HMCLs (2) KMS27 and (3) KMS28BM.

Forced MYC Activation Repressed p53-Mediated miR-34 Expression in MYC-ER Cell Lines
Because Myc inhibition enhances p53-mediated miR-34 family expression, we examined whether MYC activation could suppress p53-induced miR-34 family expression. We used an MYC-ER cell line that expresses MYC but masks its nuclear translocation signal with the estrogen receptor ligand binding domain (ER). When tamoxifen (4OHT) binds to ER, the nuclear translocation signal is unmasked, allowing Myc translocation to the nucleus and increased activity [17,18]. We simultaneously induced p53 accumulation and MYC activation in three MYC-ER cell lines, namely, osteosarcoma cell line (1) U2OS harboring WT-TP53, and HMCLs (2) KMS27 and (3) KMS28BM.

MM Cell Proliferation and Apoptosis after Co-Treatment with Nutlin-3 and Myc Inhibitor
Because Myc inhibition enhances TP53-mediated miR-34 family expression, we next examined whether p53 accumulation and Myc inhibition synergistically altered MM cell proliferation and apoptosis. In MM.1S, nutlin-3 markedly reduced cell proliferation. The Myc inhibitor alone did not affect the proliferation and did not show synergistic effects. (Figure 9A) In KMS27 and KMS28BM, neither nutlin-3 nor the Myc inhibitor alone altered the proliferation, but the combination slightly suppressed the proliferation (Figure 9B,C). Apoptosis or cell death was not significantly increased by p53 accumulation or Myc inhibition (( Figures 9A-C and S2). Because Myc inhibition enhances TP53-mediated miR-34 family expression, we next examined whether p53 accumulation and Myc inhibition synergistically altered MM cell proliferation and apoptosis. In MM.1S, nutlin-3 markedly reduced cell proliferation. The Myc inhibitor alone did not affect the proliferation and did not show synergistic effects. (Figure 9A) In KMS27 and KMS28BM, neither nutlin-3 nor the Myc inhibitor alone altered the proliferation, but the combination slightly suppressed the proliferation (Figure 9B,C). Apoptosis or cell death was not significantly increased by p53 accumulation or Myc inhibition (( Figure 9A-C and Figure S2). Forced p53 expression markedly reduced the proliferation of Tet-on p53 KMS26 and Tet-on OPM2, but the Myc inhibitor, either alone or in combination with p53 expression, did not alter the proliferation (Figure 10A,B). Apoptosis or cell death was increased by forced p53 expression but was not increased by the Myc inhibitor in both cell lines. Forced p53 expression markedly reduced the proliferation of Tet-on p53 KMS26 and Tet-on OPM2, but the Myc inhibitor, either alone or in combination with p53 expression, did not alter the proliferation (Figure 10A,B). Apoptosis or cell death was increased by forced p53 expression but was not increased by the Myc inhibitor in both cell lines. Because Myc inhibition enhances TP53-mediated miR-34 family expression, we next examined whether p53 accumulation and Myc inhibition synergistically altered MM cell proliferation and apoptosis. In MM.1S, nutlin-3 markedly reduced cell proliferation. The Myc inhibitor alone did not affect the proliferation and did not show synergistic effects. (Figure 9A) In KMS27 and KMS28BM, neither nutlin-3 nor the Myc inhibitor alone altered the proliferation, but the combination slightly suppressed the proliferation (Figure 9B,C). Apoptosis or cell death was not significantly increased by p53 accumulation or Myc inhibition (( Figure 9A-C and Figure S2). Forced p53 expression markedly reduced the proliferation of Tet-on p53 KMS26 and Tet-on OPM2, but the Myc inhibitor, either alone or in combination with p53 expression, did not alter the proliferation ( Figure 10A,B). Apoptosis or cell death was increased by forced p53 expression but was not increased by the Myc inhibitor in both cell lines.

Discussion
In this study, we demonstrated that p53 accumulation induced miR-34 family expression, an effect that was enhanced by inhibiting MYC activity. In contrast, forced MYC activation via the MYC-ER system suppressed TP53 to mediate miR-34 family expression in the MM cells. The MYC expression levels were inversely correlated with the mature miR-34 expression levels in the bone marrow plasma cells of MM and MGUS.
We found that nutlin-3, a drug that accumulates the p53 protein, increased miR-34a/b and pri-miR-34a in MM.1S with WT p53 and in some p53-mutant HMCLs but not in p53deletion HMCLs. The miR-34 family is known to be induced by p53 and by genes related to cell growth arrest and apoptosis, such as p21, MDM2, and PUMA [15]. Furthermore, nutlin-3 induces the expressions of miR-192, 194, 215 [20], p21, MDM2, and PUMA [21] in p53-WT HMCLs. However, the drug's capacity to induce miR-34 has not yet been exhibited in MM. Our current results are consistent with those of previous reports that nutlin-3 increases miR-34 expression in retinoblastoma and dopaminergic neuroblastoma cells [22,23]. The forced expression of WT p53 using the Tet-on system also induced mature miR-34a/b and pri-miR-34a expressions in all tested cell lines, indicating that WT p53 has the capacity to induce miR-34 family expression in MM.
However, we found that MM had a higher p53 expression than MGUS but a lower miR-34 family expression, suggesting a mismatch between TP53 and miR-34 in this cancer. Although frequently found in many cancer cells [24,25], deleterious TP53 mutations are rare in MM [21,22,24], meaning that TP53 dysfunction is unlikely to be causing miR-34 suppression. This discrepancy suggests the presence of factors that inhibit the p53 induction of miR-34 expression.

Discussion
In this study, we demonstrated that p53 accumulation induced miR-34 family expression, an effect that was enhanced by inhibiting MYC activity. In contrast, forced MYC activation via the MYC-ER system suppressed TP53 to mediate miR-34 family expression in the MM cells. The MYC expression levels were inversely correlated with the mature miR-34 expression levels in the bone marrow plasma cells of MM and MGUS.
We found that nutlin-3, a drug that accumulates the p53 protein, increased miR-34a/b and pri-miR-34a in MM.1S with WT p53 and in some p53-mutant HMCLs but not in p53deletion HMCLs. The miR-34 family is known to be induced by p53 and by genes related to cell growth arrest and apoptosis, such as p21, MDM2, and PUMA [15]. Furthermore, nutlin-3 induces the expressions of miR-192, 194, 215 [20], p21, MDM2, and PUMA [21] in p53-WT HMCLs. However, the drug's capacity to induce miR-34 has not yet been exhibited in MM. Our current results are consistent with those of previous reports that nutlin-3 increases miR-34 expression in retinoblastoma and dopaminergic neuroblastoma cells [22,23]. The forced expression of WT p53 using the Tet-on system also induced mature miR-34a/b and pri-miR-34a expressions in all tested cell lines, indicating that WT p53 has the capacity to induce miR-34 family expression in MM.
However, we found that MM had a higher p53 expression than MGUS but a lower miR-34 family expression, suggesting a mismatch between TP53 and miR-34 in this cancer. Although frequently found in many cancer cells [24,25], deleterious TP53 mutations are rare in MM [21,22,24], meaning that TP53 dysfunction is unlikely to be causing miR-34 suppression. This discrepancy suggests the presence of factors that inhibit the p53 induction of miR-34 expression.
Next, we simultaneously inhibited MYC while increasing miR-34 family expression by activating WT p53 with the nutlin-3 or Tet-on system. Although each HMCL had variable responses, MYC inhibition generally further increased miR-34a expression, suggesting that activated MYC represses p53-inducible miR-34.
Therefore, we investigated whether MYC suppresses p53-induced miR-34 family expression using MYC-ER cell lines that can be forced to activate MYC. In the WT TP53 osteosarcoma cell line (U2OS), MYC activation suppressed p53-induced miR-34a expression. For the two HMCLs, MYC activation reduced pri-miR-34a expression in the KMS27 cells, as well as pri-miR-34a and mature miR-34a expressions in the KMS28BM cells. Taken together, our findings support the hypothesis that MYC represses p53-induced miR-34 family expression.
Despite our results, there are still several unexplained phenomena. First, in diffuse large B-cell lymphoma, miR-34a expression is regulated in three ways: direct MYC mediation, epigenetic repression of the miR-34a promoter region, and miR-34a deletion [33]. However, the exact method of miR-34a regulation in MM is less clear. We successfully demonstrated a connection between MYC and miR-34 in plasma cells from the bone marrow of patients with MM and MGUS, specifically showing that TP53 and MYC were negatively correlated with mature miR-34 expression. Thus, MYC appears to suppress p53-induced miR-34 expressions in samples from patients and not only in the HMCL model.
We plan to perform studies using methods such as MYC ChIP assays to better understand the relationship between MYC and miR-34 in MM, clarifying whether MYC transcriptionally represses miR-34 family expression or acts via other mechanisms. While the in vitro results demonstrating this mechanism are convincing, further in vivo experiments need to be performed to demonstrate that this effect occurs in the presence of other microenvironmental mediators.
Although our results show that miR-34 expression was synergistically upregulated by p53 accumulation and Myc inhibition, the miR-34 upregulation was not translated to the suppression of proliferation or the cell death of HMCL. We used a relatively small amount of the Myc inhibitor because a larger amount greatly decreased miR-34 expression; thus, Myc inhibition did not affect cell proliferation or death. Our results might indicate that miR-34 plays roles other than affecting cell growth and survival.
In conclusion, we found that elevated WT p53 induced miR-34 family expression in MM cells, while elevated MYC suppressed miR-34 family expression. These patterns indicate that activated MYC can lead to MM development and malignant transformation because it inhibits p53-dependent miRNA expression, which functions as a tumor suppressor.