PRL-3 exerts oncogenic functions in myeloid leukemia cells via aberrant dephosphorylation of stathmin and activation of STAT3 signaling

Abstract

PRL-3, an oncogenic dual-specificity phosphatase, is overexpressed in 50% of acute myeloid leukemia patients. Stathmin has been identified as a downstream target of PRL-3 in colorectal cancer. However, the correlation between PRL-3 and stathmin in myeloid leukemia is unclear. In this study, we revealed the positive correlation between PRL-3 and stathmin in myeloid leukemia. Knockdown of the PRL-3 gene by shRNA reduced the expression of downstream stathmin, suppressed cell proliferation, induced G2/M arrest and cell apoptosis, and inhibited migration and invasion in myeloid leukemia cells. Moreover, our study was the first to provide evidence that silencing PRL-3 increased the phosphorylation level in Ser16, Ser25, Ser38, and Ser63 of stathmin, and in turn inhibited the STAT3 and STAT5 signaling in myeloid leukemia cells. This evidence points to a promoted role for PRL-3 in the progression of myeloid leukemia, and PRL-3 could be a possible new treatment target.

Keywords: PRL-3; STATs signaling; myeloid leukemia; serine phosphorylation; stathmin.

Figure 1

Expression of PRL-3 and stathmin in clinical samples and cell lines detected by western blot. (A–C) Expression of PRL-3 and stathmin in de novo myeloid leukemia patients. (D, E) Expression of PRL-3 and stathmin in myeloid leukemia cell lines. (*P<0.05, **P<0.01, vs. healthy normal control). Note: N, healthy normal control. P, de novo patient.

Figure 2

Expression of PRL-3 and stathmin was assessed after PRL-3-silencing in K562 and K562/G01 cells. (A) The mRNA expression of PRL-3 after transfection. (B) The mRNA expression of stathmin after PRL-3-silencing. (C, D) Quantification of PRL-3 and stathmin were normalized to β-actin. (E) Western blot of PRL-3 and stathmin expression were detected after shPRL-3. (*P<0.05, **P<0.01, vs. NC group).

Figure 3

Effects of PRL-3-silencing on cell proliferation and apoptosis were evaluated in K562 and K562/G01 cells. (A) A cell growth curve was plotted based on the OD value (proportional to cell numbers) obtained at different time points following transfection. (B, C) Colonies containing ≥40 cells were counted on day 7 using a microscope (×200). (D) Cells were labeled by PI and analyzed using FCM. (E) Apoptotic cells were measured by FCM. Dot plots show 7-AAD (y-axis) vs. Annexin-V (x-axis). (*P<0.05, **P<0.01, vs. NC group).

Figure 4

Effects of PRL-3-silencing on cell migration and invasion were evaluated in K562 and K562/G01 cells. (A) The OD values (proportional to cell numbers) of migrated cells were measured by MTS assay. (B) The invasion cell numbers were counted under microscope in five HP fields. (C) Wright-Giemsa stained invasion cells were observed under microscope (×200). (D, E) Quantification of MMP2 and MMP9 were normalized to β-actin. (F) MMP2, MMP9, PRL-3 and β-actin expression by western blot. (*P<0.05, **P<0.01, vs. NC group).

Figure 5

The protein phosphorylation of stathmin and STATs signaling expressed in K562 and K562/G01 cells after PRL-3-silencing. (A) Stathmin, the four stathmin-serine sites, and β-actin expression were detected by western blot. (B–E) Quantification of stathmin-phospho (Ser16, Ser25, Ser38, and Ser63) were normalized to stathmin. (F) STAT3, p-STAT3, STAT5, p-STAT5 and β-actin expression were detected by western blot. (G–J) Quantification of p-STAT3 and p-STAT5 were normalized to STAT3 and STAT5, respectively. (*P<0.05, **P<0.01, vs. NC group).

Figure 6

Suggested mechanisms for a possible interplay between PRL-3, stathmin, and STATs. With blocking of PRL-3, expression and activity of stathmin is reduced, and STAT3 and STAT5 activity are suppressed in K562 cells. The proliferation, migration, and invasion are reduced. In contrast, expression and activity of stathmin is not altered with shPRL-3 in K562/G01 cells, which results in K562/G01 cells maintaining malignant phenotypes.