Background
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma in adults, contributing to nearly 40% of new diagnoses [
1]. However, DLBCL is involuted both in clinical presentation and morphology. To resolve this problem, DLBCL is now classified as GCB-DLBCL and ABC-DLBCL by genetic profiling [
2,
3], and patients with the ABC subtype show a significantly poorer outcome compared with the GCB subtype [
4]. Immunohistochemistry (IHC) for CD10, Bcl-6 and MUM1 can also differentiate between the GCB and non-GCB subtypes of DLBCL and predicts similar outcomes to genetic profiling [
5]. Improved therapies are required for all patients with DLBCL but most urgently for those with the ABC subtype, which is the most chemoresistant and has a worse prognosis [
6].
ABC-DLBCL is associated with many different oncogenic events, but all ultimately act on nuclear factor-κB (NF-κB) to promote lymphomagenesis [
7]. Therefore, signal transducer and activator of transcription 3 (Stat3), which cooperates with NF-κB signaling, is a promising candidate for ABC-DLBCL targeted therapy [
8,
9]. Stat3 is constitutively activated in various tumor types, contributing to enhanced proliferation, survival, angiogenesis and immune evasion via several mechanisms [
10‐
14]. Constitutively activated Stat3 in ABC-DLBCL is associated with poor survival [
15]; moreover, Stat3 activation is a biomarker for poor survival in DLBCL after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment [
16].
Fbw7, also known as CDC4, is a substrate recognition element of the evolutionarily conserved SCF-type ubiquitin ligase complex. Acting as a tumor suppressor in human cancer, Fbw7 substrates include several proto-oncogenes, which are ubiquitylated and tagged for proteasomal degradation [
17‐
26]. Fbw7 genetic deletion results in developmental defects, embryonic lethality and genetic instability, and Fbw7 inactivation by loss of expression or mutation is associated with tumor development [
27]. In the hematopoietic system, Fbw7 inactivation leads to hematopoietic stem cell (HSC) depletion by active cell cycling, which can initiate leukemia [
28,
29]. Another study showed that HSC differentiation was mediated by Fbw7 regulating Myc stability [
30]. Although Fbw7 targets various substrates for degradation that have crucial roles in cell cycle, apoptosis and differentiation, the role of Fbw7-mediated degradation of such targets remains unclear.
Here, we investigate Fbw7 expression in DLBCL. Interestingly, Fbw7 showed lower expression in the non-GCB-DLBCL subtype compared with GCB-DLBCL. The major phenotype associated with Fbw7 overexpression in ABC-DLBCL cell lines was the regulation of cell apoptosis. Furthermore, we confirmed that Fbw7 targets Stat3 and pStat3Tyr705 for ubiquitin-dependent degradation., and the downstream anti-apoptosis target genes of activated Stat3, including Pim1, Survivin, Mcl-1, Myc, Bcl2 and Bcl-xl showed reduced mRNA expression following exogenous Fbw7 overexpression, in a cell-dependent manner. Together, these results reveal an Fbw7-dependent mechanism that regulates Stat3 ubiquitylation and degradation, providing new insight into the tumor suppressor role of Fbw7, and suggesting that Fbw7 may offer a promising new approach to ABC-DLBCL therapy.
Methods
Reagents
The following antibodies were used in the study: anti-Fbw7 (for IHC) (H00055294-M02, Abnova, Taipei City, Taiwan), anti-Fbw7 (for western blots) (ab109617, Abcam, Cambridge, UK), anti-Stat3, 12640; anti-phospho-Stat3Tyr705, 4113; anti-Ubiquitin, 3936 (Cell Signaling Technology, Beverly, MA, USA), anti-Myc tag, 16286–1-AP; anti-Flag tag, 66008–2-Ig; and anti-β-actin, 60008–2-Ig; anti-Myc, 10828–1-AP; anti-Notch, 10062–2-AP; anti-Jun, 10024–2-AP; anti-DEK, 16448–1-AP; anti-Mcl1, 16225–1-AP(Proteintech, Rosemont, IL, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Amresco (Dallas, TX, USA).
Cell culture
The DLBCL cell lines SU-DHL-2 and OCI-LY-3 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), and HEK293T cells were cultured in DMEM supplemented with 10% FBS. Cells were maintained in a humidified chamber with 5% CO2 at 37 °C. The identity of cell lines was confirmed by short tandem repeats-polymerase chain reaction (STR-PCR) genotyping.
Cell transfection
Vectors containing Fbw7, Stat3 and Ubiquitin were generated by cloning PCR amplified full-length human cDNAs into pcDNA3.1. And the human Stat3 siRNA target sequence was 5′-CACAT GCCAC TTTGG TGTTT CATAA-3′. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was using to perform transfections according to the manufacturer’s instructions.
Tissue samples and IHC
From 2005 to 2011, 165 human DLBCL samples were collected at the Guangdong General Hospital. The study was approved by Guangdong general hospital Biomedical Research Ethics Committee, and written informed consent was obtained from all the patients. Patients with DLBCL have been followed up for at least 5 years with a intervals of 1–3 months until July 2016. The clinicopathological characteristics of the DLBCL patients are showed in Additional files
1 and
2.
Samples were probed using the indicated antibodies. Paraffin-embedded samples were made into a tissue microarray. Staining was evaluated by two blinded individuals, and the scoring criteria was: 0 (no staining), 1 (weak staining), 2 (intermediate staining) and 3 (strong staining). Two pathologist gave a score and the final score was the average.
Apoptosis analysis
Cells were first transfected with an Fbw7 expression plasmid or vector control, and then Doxorubicin (MP Biomedicals, Illkirch-Graffenstaden France) was used to induce apoptosis. We used the Annexin V-FITC Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China), according to the manufacturer’s instructions, and the percentage of apoptotic cells was detected by flow cytometry analysis.
Cell viability assay
For cell viability assay, 5 × 104 cells per well were plated in 96-well, and then incubated with the appropriate medium containing Doxorubicin for 24 h. Assays were performed using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions.
Western blotting
Cells were lysed in RIPA lysis buffer (0.1% SDS, 50 mM Tris containing 150 mM NaCl, 1% Triton X-100 and 1% sodium deoxycholate; pH 7.2) with cocktails inhibitor of protease and phosphatase (Merck, Kenilworth, NJ, USA) on ice for 30 min and centrifuged at 14 000 × g for 30 min. According to the protein concentration of BCA Assay (Pierce, Rockford, IL, USA), 40 μg of protein was loaded on 8% SDS-PAGE gels. And then protein was transferred to PVDF membranes (Millipore, Billerica, MA, USA). Following transfer, blots were blocked, incubated with primary and secondary antibodies and exposed to film using standard procedures.
Immunoprecipitation and ubiquitination assay
Cells were lysed in RIPA lysis buffer, and the lysates were immunoprecipitated with the indicated antibodies on protein A/G beads (Millipore) overnight. The beads were then washed and boiled in SDS loading buffer. Immunoprecipitated protein complexes were assessed using Western blotting. To detect ubiquitination of Stat3 and pStat3Tyr705, 10 mM N-ethylmaleimide was added in the lysis buffer.
RNA extraction and qPCR analysis
Total RNAs were purified using RNAiso Plus, and first-strand cDNA was generated with PrimeScript RT Master Mix (Takara, Shiga, Japan). qPCR was carried out using SYBR Premix Ex Taq (Takara) on an ABI 7500 PCR system (Applied Biosystems, Carlsbad, CA, USA). The PCR protocol was made up of 40 cycles of clocking at 95 °C for 5 s and 60 °C for 30 s. The data was represented relative to β-actin, calculated using the 2
−ΔΔCT method. The primers for PCR reactions are listed in Additional file
3.
Statistical analysis
Statistical analyses were carried out using the SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Data are shown as mean ± SD. The relationships between Fbw7 expression and other clinicopathological factors were determined using Pearson χ
2 test. Kaplan-Meier survival analysis was applied to illustrate the outcome relevance of Fbw7 in univariate analysis. Each assay was performed in three repeated experiments. The Student t test was used to compare two groups of independent samples. Correlations between Fbw7 and pStat3Tyr705 levels were confirmed using the Spearman rank correlation. Values of P < 0.05 were considered significant.
Discussion
The ubiquitin-ligase Fbw7 targets several proto-oncogenes for ubiquitination and degradation and acts as a tumor suppressor in many human malignancies. However, the Fbw7 substrates that have important roles in the development of specific cancers are unknown. Here, we investigated Fbw7 in ABC-DLBCL and found that Fbw7 targets Stat3 for ubiquitylation and degradation, and that Fbw7 inhibits downstream anti-apoptotic targets of Stat3.
DLBCL is attributable for nearly 40% of all non-Hodgkin lymphoma diagnoses and has been divided into the molecular subtypes GCB and ABC by genetic profiling. Overall survival is significantly reduced in the ABC-DLBCL compared with the GCB-DLBCL. In our study, we found that Fbw7 expression was reduced in DLBCL associated with the ABC subtype (Fig.
1b). Low Fbw7 expression also correlated with a poor prognosis (Fig.
1d). We also demonstrate that Fbw7 is an apoptosis regulator through flow cytometry analysis and cell viability assays (Fig.
2b and
c). Previous studies have shown that Fbw7 targets c-Jun and Mcl-1, regulating apoptosis [
18,
20], and these data interested us to investigate its molecular mechanism for regulating apoptosis in ABC-DLBCL.
Stat3, an important transcript factor in many human cancers, shows a high level of expression and activation in ABC-DLBCL [
15]. Constitutive Stat3 activation is also a biomarker for poor prognosis after R-CHOP therapy [
16]. Previous studies demonstrated Stat3 siRNA or kinase inhibitors reduced tumor proliferation in vitro [
31]. These reports suggest targeting Stat3 could be a promising approach to therapy in ABC-DLBCL. Our data confirmed Fbw7 targets Stat3 for ubiquitylation and degradation, especially Stat3 phosphorylated at tyrosine 705. First, we found Fbw7 influenced the stability of Stat3 and pStat3
Tyr705 in a dose-dependent manner (Fig.
3b). Further, we showed that Fbw7 interacts with Stat3 and pStat3
Tyr705 in ABC-DLBCL cells by co-immunoprecipitation (Fig.
4a-c). Moreover, Fbw7 interacts with Stat3 and pStat3
Tyr705 to regulate their ubiquitylation and degradation. It has been reported that Fbw7 binds to its substrates after they have been phosphorylated within conserved phospho-degron motifs, called Cdc4 phospho-degrons (CPDs) [
32]. Several studies have demonstrated that Fbw7 targets phosphorylated substrates, including Myc, c-Jun, Mcl-1, Notch and KLF2 [
17,
18,
20‐
22]. The ability of Fbw7 to degrade these oncogenes, specifically, makes it a tumor suppressor. Our results showed that Fbw7 interacts with and degrades Stat3 in ABC-DLBCL, and therefore; may be a viable target for Stat3-directed therapy.
Stat3 is required for tumor cell proliferation, infiltration, differentiation and apoptosis inhibition. Stat3 is activated by phosphorylation, which produces a molecule that spontaneously dimerizes, binds to DNA and activates transcription of downstream target genes [
10,
11], some of which block apoptosis, including Myc, Survivin, Bcl-2, Mcl-1, Pim-1 and Bcl-xl [
33‐
36]. Our results also revealed that Fbw7 overexpression reduced mRNA levels of these target genes in a cell-dependent manner in ABC-DLBCL cell lines. There was a significant reduction in Survivin, Pim-1 and Bcl-2 mRNA levels in SU-DHL-2 cells (Fig.
5c, right), as well as a significant reduction in Myc, Survivin, Mcl-1, Bcl-2 and Bcl-xl mRNA levels in OCI-LY-3 cells (Fig.
5c, left). Thus, both our clinical and experimental data suggest Fbw7 regulates Stat3 stability and downstream signaling, making it a promising target for therapy. Although it is well known that cyclin D1 is the target gene of Stat3 which regulates cell proliferation and cell cycle, qPCR revealed that Fbw7 overexpression did not result in significant reduction of cyclin D1 (Additional file
6). And the results of CCK8 proliferation assays and cell cycle did not show significant difference after Fbw7 overexpression in ABC-DLBCL cell lines (Additional file
4). It’s reported that only 2.1% of patients in total of 1435 express cyclin D1 in DLBCL [
37]. Therefore our negative results of cyclin D1 may be related to its missing expression. Moreover, Fbw7-induced degradation of STAT3 is more important than other reported tumorigenesis including Myc, Notch, Jun, DEK and MCL1 in ABC-DLBCL (Additional file
7).
Fbw7 regulates a proliferative network that includes several oncogenes, and therefore, is considered a tumor suppressor in human cancers. There may be a viable approach for therapies directed at Fbw7 and its substrates. Studies have shown that Fbw7 expression is regulated by upstream proteins including p53, EBP2, Numb4, Pin1 and Hes-5 [
38‐
41]. In addition, multiple microRNAs, including miR-25, miR-129-5p and miR-223 have also been demonstrated to regulate Fbw7 expression [
42‐
44]. Together, these factors comprise a complex regulatory network that controls Fbw7 expression. However, exploiting this regulatory network for a means to restore or increase Fbw7 expression requires a deeper understanding of its transcriptional and post-transcriptional regulation. Further research into the upstream pathways that phosphorylate Stat3, marking it for Fbw7-mediated ubiquitylation, could also be a promising approach for drug discovery in ABC-DLBCL.
Acknowledgements
Not applicable.