MicroRNAs (miRs) are involved in tumorigenesis by regulating tumor suppressor genes and/or oncogenes. MiR187 was overexpressed in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and associated with high Ki67 expression, elevated lactate dehydrogenase, advanced International Prognostic Index and poor prognosis of patients. In vitro, ectopic expression of miR187 in T-lymphoma cell lines accelerated tumor cell proliferation, whereas treatment with miR187 inhibitor reduced cell growth. MiR187 downregulated tumor suppressor gene disabled homolog-2 (Dab2), decreased the interaction of Dab2 with adapter protein Grb2, resulting in Ras activation, phosphorylation/activation of extracellular signal-regulated kinase (ERK) and AKT, and subsequent stabilization of MYC oncoprotein. MiR187-overexpressing cells were resistant to chemotherapeutic agents like doxorubicin, cyclophosphamide, cisplatin and gemcitabine, but sensitive to the proteasome inhibitor bortezomib. Bortezomib inhibited T-lymphoma cell proliferation by downregulating miR187, dephosphorylating ERK and AKT and degrading MYC. In a murine xenograft model established with subcutaneous injection of Jurkat cells, bortezomib particularly retarded the growth of miR187-overexpressing tumors, consistent with the downregulation of miR187, Ki67 and MYC expression. Collectively, these findings indicated that miR187 was related to tumor progression in PTCL-NOS through modulating Ras-mediated ERK/AKT/MYC axis. Although potentially oncogenic, miR187 indicated the sensitivity of T-lymphoma cells to bortezomib. Cooperatively targeting ERK and AKT could be a promising clinical strategy in treating MYC-driven lymphoid malignancies.
T-cell lymphoma represents the main aggressive neoplastic disorder of T lymphocytes, generally resistant to chemotherapy.1 Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is the most frequent subtype of T-cell lymphoma and heterogeneous in morphological, clinical and molecular characteristics. Biomarkers related to the malignant behavior of tumor cells remain to be investigated and may become potential candidates for targeted therapy in PTCL-NOS.
Recent experimental data have shown that MYC is critically involved in PTCL development.2 Clinically, amplification of this proto-oncogene is frequently observed in T-leukemia/lymphoma patients.3, 4 MYC regulates proliferative pathways vital for cancer progression.5 Functioned as upstream kinases of MYC, extracellular signal-regulated kinase (ERK) and AKT can regulate MYC phosphorylation, preventing MYC from degradation and resulting in subsequent activation.6 Accordingly, overexpression of ERK and/or AKT are closely related to tumor cell proliferation and sensitivity to chemotherapy.7, 8 However, how ERK and AKT modulate MYC to induce tumor progression and chemoresistance in PTCL-NOS is not yet clearly defined.
MicroRNAs (miRs), a class of 19- to 23-nucleotide non-coding RNA molecules, regulate gene expression by targeting mRNA at the 3′untranslated region.9 Growing evidence suggested that miRs have oncogenic potential through regulating tumor suppressor genes and/or oncogenes, and are important in tumorigenesis and resistance to chemotherapy.10 More recently, it has been reported that miR187 is overexpressed and indicated poor disease outcome in solid tumors.11, 12, 13 In the present study, we assessed the miR187 expression, as well as its relation to tumor progression and drug sensitivity, in PTCL-NOS.
Patients and methods
Sixty-three patients diagnosed with PTCL-NOS and 36 patients with T-cell acute lymphoblastic leukemia were included in this study. Histologic diagnoses were established according to the WHO classification.14 Induction chemotherapy consisted of six to eight cycles of CHOP or CHOP-like regimen. Thirty-six age- and sex-matched cases with reactive hyperplasia were referred as controls. The study was approved by the institutional review board with informed consent obtained in accordance with the Declaration of Helsinki.
Cell lines and reagents
T-lymphoma cell lines Jurkat, MOLT4 and HUT78 were available from American Type Culture Collection (Manassas, VA, USA) and Karpas was from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). Bortezomib was from Millennium Pharmaceuticals (Cambridge, MA, USA). Specific inhibitor of ERK (FR180204) and AKT (Wortmannin) were from Merck KGaA (Darmstadt, Germany).
Cell proliferation assay
Cell proliferation was measured by the MTT and EdU incorporation assays. Cells were seeded in 96-well plates and incubated with the indicated concentrations of reagents at 37 °C. After 72 h incubation, 0.1 mg of MTT was added to each well and the absorbance was measured at 490 nm by spectrophotometry. An EdU assay was conducted using the Cell-Light EdU imaging kit (RiboBio, Guangzhou, China) according to the manufacturer’s instruction.
Flow cytometric analysis
Cell cycle was assessed by the distribution of nuclear DNA content. Cells were collected, washed in phosphate-buffered saline and fixed overnight in 75% ethanol at −20 °C, treated with 1% RNaseA for at least 15 min at 37 °C and stained with 50 μg/ml propidium iodide. Cell apoptosis was analyzed using the Annexin V-PE kit (BD Pharmingen, Franklin Lakes, NJ, USA).
Total RNA was extracted from 20 μm-thick paraffin (n=30) or frozen sections (n=33) using the RecoverAll total nucleic acid isolation kit (Applied Biosystem, Carlsbad, CA, USA) or Trizol agent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. MiR187 expression was analyzed by real-time quantitative RT-PCR using the miRNA reverse transcription kit, hsa-miR 187 assay and 7500HT Fast Real-time PCR system (Applied Biosystem). RNU24 was used as an endogenous control and Jurkat cells for calibration. A relative quantification was calculated using the ΔΔCT method.15
Cells were lysed in 200 μl lysis buffer (0.5 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS and 5% β-mercaptoethanol). Protein extracts (20 μg) were electrophoresed on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dried milk in Tris-buffered saline and incubated for 2 h at room temperature with an appropriate primary antibody, followed by the horseradish peroxidase-conjugated secondary antibody. The immunocomplexes were visualized using the chemiluminescence phototope-horseradish-peroxidase kit. Actin was used to ensure equivalent protein loading. Antibodies against phosphorylated-ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated-AKT (p-AKT), AKT, Actin and the chemiluminescence phototope-horseradish-peroxidase kit were obtained from Cell Signaling (Beverly, MA, USA). Antibodies against disabled homolog-2 (Dab2), growth factor receptor binding protein-2 (Grb2), serine-62 (S62) MYC, threonine-58 (T58) MYC and MYC were from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-mouse-IgG and goat anti-rabbit-IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ras-GTP was assessed in lysates by a GST-RBD pull down assay using the Ras activation assay biochem kit (Cytoskeleton, Denver, CO, USA).
Immunohistochemistry and immunofluorescence assay
Immunohistochemistry was performed on 5 μm-paraffin sections with an indirect immunoperoxidase method using antibodies against Ki67 (DAKO, Glostrup, Denmark), p-ERK, p-AKT (Cell Signaling) and MYC (Abcam). Expression levels were scored semi-quantitatively based on the percentage of positive cells: +, <25%; ++, 25–49%; +++, 50–74%; ++++, 75–100%.
An immunofluorescence assay was performed on acetone-fixed cells using rabbit anti-human-Dab2, mouse anti-human Grb2, rabbit anti-human-T58 MYC and rabbit anti-human-S62 MYC as primary antibodies (Abcam), and AlexaFluor647-conjugated donkey anti-mouse-IgG (Invitrogen), TexasRed-conjugated donkey anti-rabbit-IgG antibodies (Abcam) as secondary antibodies. Nuclei were counterstained with DAPI.
Jurkat and MOLT4 cells incubated with the pEZX-187 vector (HmiR0292-MR03) or a control vector pEZX-ct (CmiR0001-MR03, Genecopia, MD, USA) were electroporated at 210 V for 25 ms in 4-mm cuvettes using a BTX ECM 830 and replated in fresh medium for further experiments. Purified plasmids, pEZX-187 or pEZX-ct was co-transfected with pMD2.G and pSPAX2 (Addgene, Cambridge, MA, USA) into package 293T cells (ATCC, Manassas, VA, USA) using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The viral particles were collected and incubated with Jurkat cells for 4 h. The stably transfected clones were selected by green fluorescence protein.
To inhibit miR187 expression, Karpas and HUT78 cells were transfected with 150 nM inhibitor using lipofectamine 2000 (Invitorgen) for 24 h. The miR187 inhibitor miRIDIAN microRNA hsa-miR-187-3p haripin inhibitor and the negative control miRIDIAN microRNA Hairpin Inhibitor Negative Control #1 were synthesized by Thermo Fisher Scientific, Dharmacon Products (Lafayette, CO, USA).
Nude mice (5 to 6 weeks old, Shanghai Laboratory Animal Center, Shanghai, China) were injected subcutaneously with 4 × 107 stable transfected Jurkat cells into the right flank. Treatments (10 mice per group) were started after the tumor became about 0.5 × 0.5 cm in surface (day 0). The untreated group received RPMI1640, whereas the other two groups received twice weekly for two weeks doxorubicin (1.25 mg/kg) or bortezomib (0.5 mg/kg), respectively. Tumor volumes were calculated as 0.5 × a(length) × b(width).2
Overall survival time was measured from the date of diagnosis to the date of death or the last follow-up. Survival functions were estimated using the Kaplan–Meier method and compared by the log-rank test. The association between miR187 and clinicopathologic parameters was analyzed by Chi-square. Differences of miR187 expression among groups were assessed using the Mann–Whitney U test. In vitro experimental results were expressed as mean±s.d. of data obtained from three separate experiments and determined using a t-test to compare variance. P<0.05 was considered statistically significant.
MiR187 was overexpressed in PTCL-NOS and related to tumor progression
Compared with reactive hyperplasia and T-cell acute lymphoblastic leukemia, miR187 was overexpressed in PTCL-NOS (P=0.0004 and P<0.0001, respectively, Figure 1a). Among all, 55 patients had complete clinical and follow-up data. MiR187 was significantly associated with elevated lactate dehydrogenase levels and the International Prognostic Index indicating intermediate-high and high-risk (P=0.0433 and P=0.0208, respectively, Table 1). The median expression of miR187 in PTCL-NOS was 688.7. The patients with miR187 expression level over and equal to the median value were regarded as high miR187 expression, whereas those below the median value were included in the low miR187 expression. The median survival of patients in the high-miR187 expression group was 16 months, significantly shorter than those of the low-miR187 expression group (49 months, P=0.0088, Figure 1b). Notably increased Ki67 positivity was observed in the high-miR187 expression group, comparing with those in the low-miR187 expression group (P=0.0128, Figure 1c).
MiR187 promoted T-lymphoma cell proliferation
To gain insight into the biological function of miR187 in T-cell lymphoma, Jurkat and MOLT4 cells were transfected with miR187 (pEZX-187). As shown in Figure 2a, expression levels of miR187 achieved 136.3±46.1 and 7.7±2.8 after transfection, respectively. Ectopic expression of miR187 significantly accelerated T-lymphoma cell growth, comparing with the control cells (pEZX-ct). Accordingly, the percentage of G0/G1 phase cells was significantly lower in pEZX-187 cells than in pEZX-ct cells (Jurkat cells 35.3±5.5% vs 52.8±2.9%, P=0.0255; MOLT4 cells 36.5±5.6% vs 49.0±4.7%, P=0.0409). As detected by the EdU incorporation assay, EdU-positive cells were significantly increased in pEZX-187 cells (55.7±6.0%), compared with those in pEZX-ct cells (21.0±5.9%, P=0.0020, Figure 2b).
Meanwhile, Karpas and HUT78 cells were transfected with a specific miR187 inhibitor. Comparing with the negative control, the inhibitor targeting miR187 resulted in the remarkable reduction of miR187 expression and tumor cell growth, increase of G0/G1 phase cells (Karpas cells 30.1±1.5% vs 37.5±2.6%, P=0.0132, HUT78 cells 23.5±2.3% vs 29.2±1.0%, P=0.0159, Figure 2c) and decrease of EdU-positive cells (51.2±7.2% vs 34.5±6.7%, P=0.0429, Figure 2d).
No obvious difference of apoptosis was found between the two groups (Jurkat cells 3.7±0.4% vs 5.7±1.9%, P=0.1400, MOLT4 cells 12.1±3.0% vs 14.9±3.7%, P=0.3736, Karpas cells 3.6±0.6% vs 4.0±0.5%, P=0.3938 and HUT78 cells 2.9±0.8% vs 2.9±1.0%, P=0.9652, Figures 2a and c).
MiR187 stabilized MYC oncoprotein through Ras-mediated ERK and AKT activation
Further mechanic study was performed on Jurkat cells transfected with miR187. As previously reported, miR187 can negatively regulate the tumor suppressive target gene Dab2.11 On immunofluorescence assay, it was found that miR187 overexpression in Jurkat cells was related to a decrease of endogenous Dab2 that binds with the adapter protein Grb2, supporting the inhibitory effect of miR187 on Dab2 (Figure 3a). Western blot confirmed that miR187-overexpressing pEZX-187 cells exhibited lower Dab2 expression than pEZX-ct cells without change of Grb2 (Figure 3b). Dab2 interacts with Grb2 to inactivate Ras, and downstream ERK and AKT.16 In parallel with decreased Dab2 expression, increased Ras activity, as well as phosphorylation of ERK and AKT, were observed in pEZX-187 cells, with total protein levels remaining constant. MYC phosphorylated at S62 and T58, as well as total MYC, were accordingly increased (Figure 3b, left panel). In Karpas cells treated with an miR187 inhibitor, Ras activity was reduced, with decreased ERK and AKT phosphorylation, as well as MYC expression (Figure 3b, right panel). Moreover, specific ERK and AKT inhibitors abrogated miR187-induced S62 MYC and T58 MYC modulation, referring MYC activation as ERK and AKT dependent (Figure 3c).
As schematically summarized in Figure 3d, in T-lymphoma cells, miR187 could suppress Dab2 and its interaction with Grb2, bind Ras with GTP, phosphorylate ERK and AKT, resulting in the modulation of S62/T58 phosphorylation and stabilization of the MYC oncoprotein, and stimulate tumor cell proliferation.
Clinically, p-ERK, p-AKT and MYC expression were detected by immunohistochemistry in primary PTCL tumors (15 cases from the high-miR187 expression group and 15 cases from the low-miR187 expression group). High miR187 expression correlated with increased positivity of p-ERK (P=0.0103, Figure 4a), p-AKT (P=0.0153 Figure 4b) and MYC expression (P=0.0262, Figure 4c).
MiR187-overexpressing T-lymphoma cells were resistant to chemotherapeutic agents but sensitive to the proteasome inhibitor bortezomib
Jurkat cells, with or without miR187 overexpression, were cultured with chemotherapeutic agents regularly applied in lymphoma treatment. The (half-maximal inhibitory concentration) IC50 of the doxorubicin-treated pEZX-187 cells was 125.1±19.4 nM, significantly higher than that of pEZX-ct cells (80.7±1.7 nM, P=0.0169, Figure 5a). An EdU incorporation assay showed that EdU-positive cells were remarkably increased in pEZX-187 cells (27.7±5.0%), comparing with those in pEZX-ct cells (7.5±6.6%, P=0.0133). Similar results were observed in Jurkat cells treated with cyclophosphamide (IC50: pEZX-187 cells 4.6±0.2 mM, pEZX-ct cells 3.2±0.2 mM, P=0.0010), gemcitabine (IC50: pEZX-187 cells 113.0±9.6 nM, pEZX-ct cells 73.6±4.5 nM, P=0.0030) and cisplatin (IC50: pEZX-187 cells 9.4±0.6 μM, pEZX-ct cells 5.0±0.6 μM, P=0.0071, Figure 5a).
Interestingly, bortezomib, the proteasome inhibitor with anti-tumor activity in lymphoma, was able to inhibit the growth of T-lymphoma cells overexpressing miR187. IC50 was 13.4±1.6 nM in pEZX-187 cells and 22.2±4.0 nM in pEZX-ct cells (P=0.0240, Figure 5b).
Bortizomib downregulated miR187 expression both in vitro and in vivo and inhibited ERK/AKT/MYC axis
To determine whether sensitivity of T-lymphoma cells to bortezomib was related to the miR187-regulated ERK/AKT/MYC expression, miR187, ERK, AKT and MYC were assessed in Jurkat cells overexpressing miR187, untreated, or treated with doxorubicin and bortezomib, respectively. Comparing with the untreated cells, miR187 remained constant in doxorubicin-treated cells, but significantly decreased in bortezomib-treated cells. In parallel with the reduced level of miR187, p-ERK and p-AKT, as well as MYC phosphorylated at S62 and at T58 and total MYC, were downregulated by bortezomib treatment (Figure 5c). In pEZX-ct cells, p-ERK, p-AKT and MYC were downregulated by bortezomib, though to a less extent than in pEZX-187 cells (Figure 5c).
In a murine xenograft model established with subcutaneous injection of Jurkat cells, the sizes of pEZX-187 tumors were significantly larger than those of pEZX-ct tumors. Comparing with the doxorubicin treatment, bortezomib treatment particularly exhibited anti-tumor activity on pEZX-187 tumors (Figure 6a). Consistent with in vitro study, miR187 expression, proliferation index Ki67 and MYC expression, not altered in the doxorubicin group, were remarkably prohibited in the bortezomib group (Figure 6b).
In addition to genetic heterogeneity revealed by gene expression profiling,17 recent studies demonstrated that epigenetic biomarkers, especially miRs, are capable to distinguish the biological characteristics of PTCL.18, 19 Our study showed that miR187 is overexpressed in PTCL-NOS and correlated with adverse clinicopathological parameters and poor disease prognosis, indicating that dysregulation of miRNA is also involved in tumor progression in PTCL-NOS.
Expression of miR187 was associated with increased Ki67 positivity in tumor samples of PTCL-NOS patients. Also in cellular transfection models of T-lymphoma, significantly increased EdU-positive tumor cells and decreased G0/G1 phase cells were observed in those overexpressing miR187. These results suggested that miR187 may function as a stimulator of tumor cell growth. This is in accordance with a previous study on ovarian cancer that miR187 provokes tumor cell proliferation and could also explain why overexpression of miR187 is related to elevated lactate dehydrogenase levels and high-risk International Prognostic Index stratification at diagnosis in PTCL-NOS patients.
Tumor suppressor gene Dab2 inhibits tumor cell growth in solid tumors.11, 16 Dab2 can modulate ERK and AKT pathways through binding to the adapter protein Grb2 and activating Ras.16, 20 In T-lymphoma cells, miR187 significantly downregulated Dab2 expression, which in turn activated ERK and AKT. Consistent with thyroid cancer, miR187 is selectively upregulated in tumors harboring RET/PTC rearrangement,13 the subtype with activation of ERK and AKT cascade.21 Consequently, ERK and AKT, as the upstream kinases of MYC oncoprotein, modulate MYC phosphorylation at the site of S6222 and T58,23 altering stabilization of the MYC protein. Therefore, as a recent report on multiple myeloma indicating that MYC could be regulated by miR126,15 miR187 is functionally relevant in T-cell lymphoma progression through Ras/ERK/AKT-mediated MYC activation.
ERK and AKT are commonly involved in tumor chemosensitivity.24 MYC is also a hallmark of aggressive, chemoresistant tumors.25, 26 MiR187 predicted poor disease outcome in patients with breast cancer and confers increased invasive potential of tumor cells.12 Owing to its effect on activating ERK and AKT, as well as MYC oncoprotein, miR187 was also linked to multiple chemoresistance of T-lymphoma cells and thereby correlated with decreased survival time of patients with PTCL-NOS. Interestingly, inhibition of ERK and AKT sensitize tumor cells to the chemotherapeutic agents.27, 28 As MYC is difficult to be overcomed by chemotherapy, even combined with immunotherapeutic agents rituximab29 or eparatuzumab,30 bio-therapeutic approaches modulating the upstream cascades like ERK and AKT are of great promise in targeting MYC oncoprotein.
Bortezomib, the first proteasome inhibitor used in the clinic, has shown clinical efficiency in PTCL.31 Our results showed that bortezomib inactivated ERK and AKT, subsequently reduced S62 MYC, T58 MYC and total MYC expression, indicating that MYC is another important target of bortezomib. Also reported in prostate cancer,32 inactivating ERK/AKT/MYC axis contributes to an alternative mechanism of action of bortezomib. Of note, response to bortezomib is in parallel with downregulation of miR187, confirming the involvement of miRs in the action of bortezomib on tumor cells.33 It has been recently reported that cMyc/miR-125b-5p signaling determines the sensitivity of bortezomib in cutaneous T-Cell lymphoma.34 Therefore, in addition to targeting the proteasome directly, bortezomib alters the expression of specific miRNAs that target downstream pathways critical for proliferation and chemoresistance. How miR187 is regulated by bortezomib remains unclear, but it provides information on the clinical efficacy of bortezomib in PTCL.
In conclusion, our findings confirmed the oncogenic potential of miR187 in PTCL-NOS. Although related to tumor progression and multiple resistance to chemotherapy, miR187 indicated the sensitivity of T-lymphoma cells to bortezomib. Targeting MYC oncoprotein by modulating ERK and AKT signaling pathways represents a promising clinical strategy in treating MYC-driven lymphoid malignancies.
This work was supported, in part, by the National Natural Science Foundation of China (Distinguished Young Scholars, 81172254 and 81101793), the Shanghai Commission of Science and Technology (11JC1407300 and 09411963000), the Program of Shanghai Subject Chief Scientists and the “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (09SG21).
About this article
Journal of Molecular Medicine (2017)