NPM1Mutations Regulate The Invasive Phenotype Via RAS/MEK/ERK Signaling In Human Leukemia Cells In Vitro

PARTⅠMutated NPM1gene regulate the expression of matrixmetalloproteases (MMPs) and angiopoietins（Ang）Mutations of nucleophosmin (NPM1) gene are the most frequentgenetic alteration in acute myeloid leukemia, which were involved inleukemogenesis. The patients with mutated NPM1frequently containextramedullary infiltration. To explore the effect of mutated NPM1gene onthe expression of matrix metalloproteases (MMPs) and angiopoietins (Ang)that associated with leukemia invasion. PINCO-flag-NPM1mA vector withNPM1mutant A gene (NPM1mA) was transfected into the leukemicTHP-1cells. The THP-1cells stably expressing NPM1mA protein (THP-1mA cells) were used in followed experiments. Invasion assays wereperformed to evaluate the invasive phenotype of THP-1cells in vitro;Gelatin zymography were used to determine the activity of MMP-2andMMP-9in culture medium; The mRNA expressions of Ang-1and Ang-2 were assayed by quantitative Real-Time PCR (qRT-PCR). The result of cellinvasion assay suggested that the introduction of NPM1mA genesignificantly enhanced the invasive potential of THP-1leukemia cells(p<0.01). Gelatin zymography showed that activity of gelatinase (MMP-2and MMP-9) in culture medium of THP-1mA cells was enhancedobviously. Moreover, The mRNA expression of Ang-1was remarkablyincreased, while the mRNA expression of Ang-2was significantlydecreased in THP-1mA cells. These results demonstrate that mutated NPM1gene promote THP-1leukemia cell invasion by enhancing the activity ofgelatinase and the expression of Ang-1, also by depressing the expression ofAng-2. PARTⅡ The effect of RAS/MEK/ERK signaling on the invasivephenotype of leukemia cells with NPM1mutationPrevious study showed that mutated NPM1gene could promoteleukemia cells invasion. To explore the key role of signal pathway in thecell invasion that mutated NPM1gene involved. Co-immunoprecipitation（IP） were performed to detect the interactionbetween NPM1mAproteinand K-RAS protein. THP-1mA cells were treated with selective inhibitors of ERK, JNK, p38MAPK and AKT. The mRNA expression ofMMP-2/MMP-9and Ang-1/Ang-2were assayed by qRT-PCR.Furthermore, Western blot and Gelatin zymography were performed todetermined the protein and activity of MMPs respectively.We demonstratedthat NPM1mA protein could interact with K-RAS protein. Moreover, theinvasive potential of THP-1mA cells was partly inhibited by ERK inhibitor(PD98059,40μM). But others pathway inhibitors (JNK inhibitor, p38MAPK inhibitor and AKT inhibitor) had not notable effect on the invasivepotential of THP-1mA cells. The mRNA and protein expression of MMP-2and MMP-9were significantly depressed in THP-1mA cells that treatedwith ERK inhibitor (PD98059). Gelatin zymography showed that theactivity of MMP-9in culture medium of THP-1mA cells treated with ERKinhibitor was repressed obviously. Furthermore, the mRNA expression ofAng-1and Ang-2were increased in THP-1mA cells treated with PD98059.These results showed a novel role of mutated NPM1gene in regulatinginvasion phenotypes of leukemia cells, and provide the evidence thatRAS/MAPK/ERK signaling are involved in this process through regulatingMMPs and Ang expression.