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Oncotarget
2015 Oct 06;630:29573-84. doi: 10.18632/oncotarget.4998.
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Stathmin 1 inhibition amplifies ruxolitinib-induced apoptosis in JAK2V617F cells.
Machado-Neto JA
,
de Melo Campos P
,
Favaro P
,
Lazarini M
,
da Silva Santos Duarte A
,
Lorand-Metze I
,
Costa FF
,
Saad ST
,
Traina F
.
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The JAK/STAT pathway is constitutively activated in myeloproliferative neoplasms and can be inhibited by ruxolitinib, a selective JAK1/2 inhibitor. The JAK2(V617F) mutation leads to constitutive STAT3 phosphorylation and potentially leads to inhibition of Stathmin 1 activity via STAT3. In support of this hypothesis, we found that, in HEL JAK2(V617F) cells, ruxolitinib treatment decreased STAT3 and Stathmin 1 association, induced Stathmin 1 activation and microtubule instability. Silencing of Stathmin 1 significantly reduced cell proliferation and clonal growth, and increased apoptosis induced by ruxolitinib. Stathmin 1 silencing also prevented ruxolitinib-induced microtubule instability. To phenocopy the effect of Stathmin 1 inhibition, cells were treated with paclitaxel, a microtubule-stabilizing drug, in association or not with ruxolitinib; combined treatment significantly increased apoptosis, when compared to monotherapy. Notably, Stathmin 1 mRNA levels were highly expressed in CD34(+) cells from primary myelofibrosis patients. We then proposed that an undesired effect of ruxolitinib treatment may constitute Stathmin 1 activation and microtubule instability in JAK2(V617F) cells. Induction of microtubule stability, through Stathmin 1 silencing or paclitaxel treatment, combined with ruxolitinib could be an effective strategy for promoting apoptosis in JAK2(V617F) cells.
Figure 1. Ruxolitinib treatment induces microtubule instabilityA. Immunoprecipitation (IP) with anti-STAT3 and immunobloting (IB) with anti-Stathmin 1 in total extracts from HEL cells, treated or not with ruxolitinib (300 nM). Isotype IgG antibody was used as a negative control of the immunoprecipitation; total cell extracts were used as positive controls for immunoblotting. B. Western blot analysis for Stathmin 1 serine 16 phosphorylation (an inhibitory site) (p-Stathmin 1S16) and alpha-tubulin acetylation (a marker of microtubule stability) (Ac-α-TubulinL40) levels in total cell extracts from HEL cells treated or not with different concentration of ruxolitinib (100 and 300 nM). The antibodies used for immunoblotting (IB) are indicated. The decreased p-Stathmin 1 and Ac-α-Tubulin levels indicate Stathmin 1 activation and microtubule instability upon ruxolitinib treatment. Immunoblotting for phospho STAT3 and total STAT3 confirmed the inhibitory effect of ruxolitinib on STAT3 activation; actin was used as a loading control. C. Confocal analysis of HEL cells, treated or not with different concentration of ruxolitinib (100 and 300 nM), displaying α-Tubulin (green) and DAPI (blue) staining; MERGE shows the overlapped images. Scale bars are shown in the figure (10 μm). Note more diffuse microtubule networks in ruxolutinib-treated cells.
Figure 2. Stathmin 1 silencing reduces cell proliferation and clonogenicityA. Stathmin 1 mRNA and protein expression in HEL cells transduced with lentivirus-mediated shRNA control (shControl) or Stathmin 1 (shSTMN1). The antibodies used for immunoblotting (IB) are indicated. B. Cell viability was determined by MTT assay after 48 hours of incubation of shSTMN1 and normalized by the corresponding shControl cells. Results are shown as mean ± SD of six independent experiments; *p < 0.05, **p < 0.01; Mann–Whitney test. C. Ki-67 mean of fluorescence intensity (M.F.I.) was determinated by flow cytrometry after incubation of shSTMN1 for 48 h and normalized by the correponding shControl cells. Results are shown as mean ± SD of four independent experiments; *p < 0.05, Student t test. D. Colonies containing viable cells were detected by MTT after 10 days of culture of shSTMN1 and normalized by the corresponding shControl cells. Colony images are representative of one experiment and the bar graphs show the mean ± SD of at least six independent experiments; ***p < 0.0001; Student t test. The assays were performed in the presence or not of ruxolitinib (100 and 300 nM) as indicated.
Figure 3. Stathmin 1 inhibition increases the pro-apoptotic effects of ruxolitinib treatmentA. Apoptosis was detected by flow cytometry in HEL cells transduced with shControl and shSTMN1 using Annexin-V/PI staining method and a representative dot plot is illustrated. B. Bar graphs show the mean ± SD of six independent experiments; **p < 0.01; Student t test. The assays were performed in the presence or not of ruxolitinib (100 and 300 nM) as indicated.
Figure 4. Stathmin 1 silencing prevents microtubule instability induced by ruxolitinib treatmentWestern blot analysis for p-Stathmin 1S16, Stathmin 1, alpha-tubulin acetylation (Ac-alpha-tubulinL40), p-STAT3Y705, p-JAK2Y221, caspase 3 (total and cleaved) and cleaved PARP1 levels in total cell extracts from shControl and shSTMN1 cells treated or not with ruxolitinib at 100 or 300 nM; membranes were reprobed with the antibody for detection of the respective total protein or actin, and developed with the ECL Western Blot Analysis System.
Figure 5. Paclitaxel-induced microtubule stability improves ruxolitinib responseA. Apoptosis was detected by flow cytometry in HEL cells treated or not with paclitaxel (5 or 10 nM) and/or ruxolitinib (300 nM) using Annexin-V/PI staining method and a representative dot plot is illustrated. B. Bar graphs show the mean ± SD of six independent experiments; *p < 0.01 vs. untreated cells, #p ≤ 0.001 vs. ruxolitinib or paclitaxel monotherapy at the corresponding dose; Student's t test. C. Cell viability was determined by MTT assay after 48 hours of incubation of HEL cells treated or not with paclitaxel (5 or 10 nM) and/or ruxolitinib (300 nM), and normalized by untreated HEL cells. Results are shown as mean ± SD of six independent experiments; *p ≤ 0.002 vs. untreated cells, #p ≤ 0.02 vs. ruxolitinib or paclitaxel monotherapy at the corresponding dose; Mann–Whitney test.
Figure 6. Paclitaxel treatment leads to Stathmin 1S16 phosphorylation and potentiates ruxolitinib-induced caspase 3/PARP1 cleavageWestern blot analysis for p-Stathmin 1S16, alpha-tubulin acetylation (Ac-alpha-acetylationL40), p-STAT3Y705, p-JAK2Y221, caspase 3 (total and cleaved) and cleaved PARP1 levels in total cell extracts from HEL cells treated, or not, with paclitaxel (5 or 10 nM) and/or ruxolitinib (300 nM); membranes were reprobed with the antibody for the detection of the respective total protein or actin, and developed with the ECL Western Blot analysis system.
Figure 7. Stathmin 1 mRNA levels in CD34+ cells from healthy donors and patients with myeloproliferative neoplasmsA. qPCR analysis of Stathmin 1 (STMN1) mRNA expression in peripheral blood (PB) CD34+ cells from healthy donors (HD), and from patients with a diagnosis of essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), and from myeloproliferative neoplasms (MPN) patients stratified by B. JAK2V617F or C. CALR exon 9 mutational status. Horizontal lines indicate medians. The number of subjects and p values (Mann–Whitney test) are indicated in the graph. D. Potential model for Stathmin 1 function in HEL cells; the constitutive activation of JAK2 by the V617F mutation leads to STAT3 phosphorylation, Stathmin 1 inhibition and microtubule stability. Ruxolitinib treatment inhibits JAK2 and STAT3 activity, decreasing the Stathmin 1 and STAT3 association, which consequently releases Stathmin 1 and leads to microtubule instability. Paclitaxel treatment overcomes the effect of ruxolitinib treatment, inhibiting Stathmin 1 and increasing microtubule stability. Abbreviations: Ac: acetylation.
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