Abstract
Nucleolin (NCL) is an important nucleocytoplasmic multifunctional protein.It plays multiple functions in cell proliferation, apoptosis and angiogenesis. NCL displays oncogenic roles in a large variety of cancer cells, which was reported as high expression in most cancers. In these cancer cells, NCL overexpression influences cell survival, proliferation and invasion through its action on different cellular pathways. However, less research on the roles of NCL in hematologic malignancies was noted. Our previous studies showed that overexpression of NCL is associated with worse prognosis in the patients with acute leukemia and NCL expressed higher in resistant HL-60/ADR than in sensitive HL-60 cells. The potential mechanisms of NCL in chemo-resistance have yet to be investigated. Here we presented the association of NCL with clinical prognosis of acute lymphoblastic leukemia (ALL) and the role of NCL in drug resistance in T-ALL lines. Clinically, the results showed that overexpressed NCL at both mRNA and protein level was associated with a poorer overall survival (OS) and relapse free survival (RFS), as an independent prognostic marker in ALL. mRNA level of NCL in de novo ALL was quantitatively higher than in complete remission(CR) status, and refractory/relapse ALL had the highest level, which relative levels using 2-∆∆Ct method were 1.386, 0.682, and 2.159 respectively (Median(QR), P <0.05) . Expression of NCL protein was observed in 58.3% (35/60) de novo samples, 8.0% (7/87) CR samples and 27.7% (13/47) refractory/relapse samples (P <0.05). Two-year OS rates ( Figure 1 ) were respectively 33.3%±8.6% vs 4.0%±1.0% for patients with NCL- negativity and NCL-positivity (P <0.05). Two-year RFS rates were respectively 16.3%±7.2% vs 3.0%±1.0% for patients with NCL-negativity and NCL-positivity (P <0.05). Upon above clinical data, we farther investigated the mechanism(s) by which NCL regulates cellular drug resistance in T-ALL Jurkat cell line. We established cell models with NCL different expression which was transfectedby lentivirus-mediated NCL overexpression vector or siRNA vector, in order to produce NCL overexpression and NCL knockdown models for comparison drug sensitivity. The IC50 of adriamycin on Jurkat cells with NCL-upregulation and NCL- downregulation were 1.362±0.271μg/ml and 0.077±0.010μg/ml respectively (P <0.05). The results of intracellular drug accumulation and effluxion analysis further strengthened the unambiguous promotion impact of NCL on insusceptibility to anticancer agent. Furthermore, we found that Ras/Raf/MEK/ERK signaling and its downstream cell apoptosis factors including c-Myc, Bcl-2 and drug resistance-associated molecules BCRP and MRP1 were all significantly up-regulated after NCL overexpression, conversely, down-regulated after NCL knockdown. Overexpressed NCL up-regulated c-Myc, Bcl-2, BCRP, LRP, MRP1 at mRNA levels, with 2-∆∆Ct values were 7.40±0.40, 1.43±0.11, 2.88±0.07, 2.13±0.02, and 3.35±15, respectively. On the contrary, knockdown of NCL led to reversed results, 2-∆∆Ct values of them as following: 0.24±0.03, 0.32±0.09, 0.03±0.01, 0.69±0.04, 0.55±0.08. Protein levels were in accordance with mRNA level. Notably, co-IP assays confirmed the NCL-Ras, NCL-ERK, NCL-BCRP and NCL-MRP1 interaction. When Ras/EKR signaling was inhibited, levels of BCRP, MRP1 were both decreased, but did not affect to NCL expression. In summary, our findings revealed the novel understanding role of NCL in ALL treatment unresponsiveness and the emerging marker of NCL as a survival predictor and treatment target in ALL.
No relevant conflicts of interest to declare.
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