Silence of MCL-1 upstream signaling by shRNA abrogates multiple myeloma growth

Objectives Multiple myeloma (MM) is an incurable B-cell cancer with accumulated clonal abnormal plasma cells in bone marrow of patients. MCL-1 (myeloid cell leukemia sequence 1) protein is an anti-apoptotic molecule in MM cells and regulated by pro-inflammatory cytokine IL-6 and downstream signaling molecules STAT3, PI3K and MAPK. The purpose of this study is to investigate the effect of STAT3, PI3K and MAPK gene silence on MCL-1 expression in human MM cells and the consequence of cell survival. Methods Lentivirus small hairpin RNA (shRNA) interference techniques were utilized to knock down STAT3, PI3K or MAPK genes. Gene and protein expression was quantified by quantitative real-time PCR and Western Blot. MM cell apoptosis was examined by annexin-V FITC/propidium iodide staining. Results Efficient silence of STAT3, PI3K, MAPK1 or MAPK2 gene robustly abrogated IL-6 enhanced MCL-1 expression and suppressed MM cell growth. Silencing STAT3 gene inhibited PI3K expression, silencing PI3K markedly abrogated STAT3 and MAPK production. Inhibition of MAPK2 gene by shMAPK2 suppressed STAT3, PI3K and MAPK1 expression in the cells. Silencing of STAT3, PI3K and MAPK2 together completely blocked MCL-1 expression in MM cells. Conclusion There is a syngeneic effect among the three independent STAT3, PI3K and MAPK2 survival-signaling pathways related to MCL-1 expression in MM cells. shRNAs silencing of STAT3, PI3K and MAPK2 together could provide an effective strategy to treat MM.


Background
Multiple myeloma (MM) is a B-cell blood cancer that characterized with excessive clonal abnormal plasma cells in the bone marrow [1]. It is the most common hematological malignancy in USA. As a B-cell lymphoma-2 (BCL-2) family member, myeloid cell leukemia-1 (MCL-1) plays critical roles in promoting the survival of MM cells [2] as well as a wide range of other cancer cells due to its anti-apoptosis activities [3]. The mechanism by which Mcl-1 blocks the progression of apoptosis is through binding and sequestering the pro-apoptotic BH3-only proteins Bim, PUMA, Noxa, Bak, and Bax [4], preventing pore formation on mitochondrial membrane of MM cells and the release of cytochrome c into the cytoplasm. In clinical and preclinical studies, pan BCL-2 protein inhibitors have been used to treat patients with multiple myeloma [5]. Those inhibitors are small synthetic BH3 mimetic molecules with the potent capability to bind to anti-apoptotic factors. Monoclonal antibodies targeting the malfunctioned plasma cells have been widely used to treat MM patients either alone or combined with chemotherapy [6].
MCL-1 is a short half-life protein. It is overexpressed in cancers cells, which contributes to drug resistance to conventional chemotherapy [7,8]. The expression of MCL-1 in MM cells is tightly regulated by survival signaling triggered by cytokines and growth factors in bone marrow microenvironment [2,9,10]. IL-6 is a key pro-inflammatory cytokine that activates MCL-1 survival signaling upstream molecules including signal transducer and activator of transcription-3 (STAT3), phosphatidylinositol-3 (PI3K), and Mitogen-activated protein kinase (MAPK) [11]. Those molecules modulate three independent signaling pathways that control MCL-1 transcription [2]. Thus, targeting IL-6 triggered MCL-1 modulating molecules offers potential therapeutic approach to treat multiple myeloma. In this study, we investigated the effects of STAT3, PI3K and MAPK RNA interference on IL-6 induced MCL-1 expression in human MM cells and the apoptotic death of the cells.

Gene silencing
STAT3, PI3K and MAPK targeting oligonucleotide sequences were chemically synthesized, used for the cloning of shRNA-encoding sequences, and inserted into a lentiviral expression vector pGV217 (GeneChem, Shanghai, China).

Quantitative Real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells as previously described [12]. PrimeScript® RT reagent Kit and SYBR® Premix Ex Taq™ II kit were obtained from TAKARA (Dalian, China) and used for conversion of total RNA to cDNA and for the detection of mRNA expression. Each PCR reaction mixture (20 μl) included 10 μl of SYBR Green Master Mix, 0.4 μl of sense and anti-sense primers, and 10 ng of cDNA. The PCR reaction mix was first denatured at 95°C for 10 minutes. PCR was then run for 40 cycles: denaturation at 95°C for 15 seconds and annealing at 60°C for 1 minute. The primer sequences for qRT-PCR were purchased from AuGCT Corporation as listed in Table 1. According to the manufacturer's instructions, the fold change with 2 -ΔΔCt method was used in qRT-PCR data analysis and β-actin was served as a control. All reactions were run in triplicate using the IQ-5 Real-Time PCR System (Bio-Rad, USA).

Cell apoptotic assay
U266 or 8266 cells (10 6 cells/well) were cultured in 12-well plates in triplicate and transfected with targeted shRNA against STAT3, PI3K, MAPK1 or MAPK2, or control shRNAs vectors for 48 h following the manufacturer's instructions. Apoptotic and viable cells fractions were assessed with annexin-V FITC/propidium iodide staining (Invitrogen, USA) on a flow cytometer (Becton, USA) following the manufacturer's instruction. The apoptotic populations were determined by ModFit software. Alternatively, The cells were harvested and washed in PBS, followed by fixation with ice-cold ethanol overnight. The cells were then washed in PBS and incubated in 1 ml staining solution (20 ug/ml propidium iodide and 10 U/ml RNaseA) for 30 min at room temperature. The cell cycle distributions were assayed by fluorescence-activated cell sorting using a flow cytometer.

MTT assay
Cell growth was assessed with the 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The cells were seeded into wells of a 96-well plate and infected with lentivirus carrying shRNA for 24 and 48 hours at 37°C. The cells were then treated with MTT solution (15 μl/well) for 4 hours at 37°C. After gentle removal of culture supernatant, the cell pellets were dissolved with 200 μl of Dimethyl sulfoxide (DMSO). Optical density (OD) at 570 nm was read on a FLUOstar OPTIMA machine (BMG Labtech). Experiments were performed in triplicate.

Western blot
U266 or 8266 cells were harvested 24 or 48 hours after IL-6 treatment and washed with cold PBS. Total proteins extracted from the cells using a RIPA cell lysis buffer (Wolsen, China) were separated on 10% SDS polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membrane (Millipore, USA). The membranes were treated with mouse anti-STAT3, rabbit anti-MCL-1, anti-PI3K or anti-MAPK1/2 antibodies, rabbit Table 1 Primer sequences for qRT-PCR anti-pSTAT-3, anti-pMAPK1/2 antibodies, and mouse anti-β-actin antibodies (1:1000 dilution) (Cell Signaling Technology, USA). The membranes were then reacted with the HRP-conjugated secondary antibodies before subjected to enhanced chemiluminescent (ECL) detection on an ECL machine (Pierce, USA). The blots were scanned and the band density was measured on the Quantity One imaging software.

Statistical analysis
All data were presented as mean ± SD (standard deviation). The MCL-1 protein expression levels post IL-6 treatment, cell growth, and percentage of apoptotic cells in different shRNA silencing were analyzed by One-way ANOVA. P < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01).

IL-6 stimulation enhanced MCL-1 expression and myeloma growth
To evaluate the effect of pro-inflammatory cytokine IL-6 on MCL-1 expression and myeloma growth, human U266 and 8226 multiple myeloma cells were treated with or without recombinant cytokine IL-6 in vitro. Total RNA and protein were isolated from the treated cells. qRT-PCR assay revealed that IL-6 treatment significantly enhanced MCL-1 gene expression in both U266 and 8226 cells after 48-hour culture compared to the control without treatment (p < 0.05) ( Figure 1A); there was a slight increase of MCL-1 in the 24-hour treatment of IL-6, but not significant. Western blot with anti-human MCL-1 monoclonal antibodies showed that IL-6 treatment (48 hours) significantly enhanced MCL-1 protein expression levels ( Figure 1B), with a 4 fold increase in U266 cells and a 2.5 fold increase in 8226 cells in comparison with no IL-6 treatment control or 24 hours treatment (p < 0.01) ( Figure 1C). While, MCL-1 levels showed no difference between 24-hour and no IL-6 treatment. Consistently, IL-6 treatment significantly enhanced the growth of myeloma cells after 48-hour treatment with IL-6 in U266 cell line ( Figure 1D) and 8226-cell line, in comparison with the treatment without IL-6 (p < 0.05) ( Figure 1E).

Interactions among STAT3, PI3K and MAPK signaling on IL-6 induced MCL-1 gene expression in myeloma cells
To explore the potential interactions among the three signaling molecules on MCL-1 expression in IL-6 treated myeloma cells, the protein expressions of STAT3, PI3K, MAPK and MCL-1 was profiled by Western blot in human 8226 myeloma cells transfected with shSTAT3-, shPI3K-, shMAPK1-or shMAPK3-bearing lentivirus. In comparison with control shRNA, silencing STAT3 protein expression by shSTAT3 completely blocked PI3K protein expression and markedly reduced p-ERK1/2, consequently suppressed the expression of the downstream MCL-1 protein in the IL-6 treated 8226 cells. However, no inhibitory effect was observed on total MAPK protein expression ( Figure 3A). We also observed that silencing PI3K with shPI3K significantly reduced the protein levels of total STAT3, pSTAT3, total MAPK, the phosphorylation of MAPK p44, and downstream MCL-1 ( Figure 3B). Compared with control shRNA treatment, silencing MAPK1 with shMAPK1 robustly suppressed MCL-1 expression, but did not affect the protein level of STAT3 and PI3K ( Figure 3C). When silencing MAPK2 expression in 8226 cells, there was a remarkable decrease of STAT3 protein expression in treated cells in comparison with shRNA control group ( Figure 3D). There was also a slight reduction of PI3K expression in shMAPK2 treated 8226 cells. Moreover, silencing STAT3, PI3K and MAPK2 at the same time completely blocked STAT3, PI3K, MAPK2 expression, with little MAPK1 expression, leading to the absence of MCL-1 production in MM cells ( Figure 3E). Taken together, there was an interaction among STAT3, PI3K and MAPK2 signaling pathways.

STAT3, PI3K and MAPK RNA interference abrogated myeloma cell growth
To analyze the effect of STAT3, PI3K and MAPK gene silencing on the apoptosis of targeted myeloma cells, IL-6 stimulated human U266 or 8226 cells were harvested 48 hours after shRNA-bearing lentivirus treatment. The cells were stained with annexin-V and propidium iodide. Flow cytometry analysis showed that silencing STAT3, PI3K, or MAPK1 or MAPK2 significantly promoted cell apoptosis in U266 cells as well as in 8226 cells, compared to control shRNA treatment (p < 0.05) ( Figure 4A and 4B). In U266 cells, targeted shRNA treatment induced an average of 44-49% cell apoptosis. While control shRNA group only showed a 33% cell apoptosis. In 8226 cells, shSTAT3, shPI3K and shMAPK treatments led to a 23-26% cell apoptosis compared with only a 13% cell apoptosis in the shRNA control group (p < 0.05). Consequently, targeted shRNA treatments markedly inhibited the growth of myeloma cells in both cell lines in comparison with control shRNA treatments ( Figure 4C and D).

Discussion
The findings in this study indicated that RNA silence of STAT3, PI3K, or MAPK gene using shRNA interference robustly inhibited MCL-1 expression in IL-6 treated human MM cells, leading to remarkable myeloma cell apoptosis. MCL-1 plays critical roles in cancer cell survival and drug resistance against chemotherapy [8,14,15] and become a promising therapeutic target for MM treatment [16]. RNA silencing/interference is a technique that can specifically and efficiently knocks down target genes. STAT3, PI3K and MAPK are three key upstream signaling molecules that control MCL-1 expression. It has showed selective targeting MCL-1 or its upstream modulatory molecules by RNA interference induced cancer cell death [17][18][19][20]. Our study extends the previous studies using shRNA gene interference technique for targeting oncogenes in MM cells. To our knowledge, it is the first report showing that efficient knockdown of PI3K or MAPK gene by shRNA could sufficiently block MCL-1 expression in MM cells and resulted in consequent cell death, which offers a potential to utilize shRN/RNA interference as an alternative of chemotherapy for treat drug-resistant MM treatment. RNA interference technique has been widely used to investigate the functional interaction among related genes by selective silence of one gene. In this study, we also assessed the mutual effect of shRNA silencing among STAT3, PI3K and MAPK genes. It was reported that STAT3 had functional activities in PI3K-driven oncogenic transformation and there was a crosstalk between STAT3 and PI3K signaling pathways in driving the transformation of murine glioblastoma [21] and lymphoblastic B-cell lymphomas [22], and caused drug resistance [23]. Qiang et al. reported that there is a crosstalk between PI3K and MAKP pathways in MM cells [24]. Consistent with their study, we found that silencing MAPK2 but not MAPK1 had marked suppressive effect on PI3K as well as STAT3. MAPK1/2 signaling network involves many cross-talk nodes and routes interactive with other cell survival signaling pathways such as PI3K [25], it is not surprising that silencing MAPK1 and MAPK2 could have distinct effects on PI3K and STAT3 expression in MM cells. Collectively, our data demonstrated that knockdown one of STAT3, PI3K and MAPK2 genes likely affected the expression of the other two genes in human MM cells and support the evidence that blockade of both PI3K and MAPK pathways caused a significantly higher percentage of human primary MM cell death than individual inhibition [26]. These data suggest that combined targeting IL-6-activated STAT3, PI3K and MAPK molecules or signaling pathways tend to provide better clinical treatment for MM patients, considering the heterogeneous phenotypes of MM and the emerging drug resistance in the course of chemotherapy [27].
In conclusion, we have efficiently delivered lentivirus shRNA to knock down STAT3, PI3K, MAPK genes and downstream MCL-1 expression in human MM cells, and increased cell apoptosis. Selectively silence of MCL-1 upstream signaling molecules STAT3, PI3K and MAPK by shRNA provides a potent strategy to treat drugresistance human MM.