Oncogenic Wip1 phosphatase is inhibited by miR-16 in the DNA damage signaling pathway

Abstract

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage-responsive proteins and reverses DNA damage-induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis.

Figure 1

miR-16 suppresses Wip1 expression by targeting WIP1 3’UTR. (A) Predicted miRNA sequences and their putative recognition sites within 3’ UTR of WIP1. (B) Wip1 expression is suppressed by ectopic miR-16 transfection. U2OS cells were transfected with the indicated miRNAs. 72 h after transfection, Wip1 protein levels in each sample were detected by immunoblotting (upper panel), quantitated according to the intensity of Wip1 bands, and normalized with the control sample (bottom panel). (C) Immunoblots of endogenous Wip1 in U2OS cells transfected with control miRNA, miR-16 or antagomir-16, and treated with 500 ng/ml NCS at 72 h after transfection. (D) MiR-16 specifically targets the 3’ UTR of WIP1. A luciferase construct containing wildtype WIP1 3’UTR (left panel) or mutant WIP1 3’ UTR (right panel) was transfected into U2OS cells with the indicated miRNAs or antagomirs. Renilla luciferase activities were measured 48 h after transfection and normalized to firefly luciferase. Relative luciferase activity (luminescence) was obtained after normalizing with the control number.

Figure 2

miR-16 inhibits the DNA damage-mediated induction of Wip1. (A) Wip1 protein is induced in response to DNA damage. U2OS cells were treated with 500 ng/ml NCS and cell lysates were harvested at indicated time points after NCS treatment. Protein levels were determined by immunoblotting. (B) Induction of Wip1 protein has a delayed onset compared to the induction of Wip1 mRNA. Levels of Wip1 mRNA and protein in the above NCS-treated cells were determined by quantitative RT-PCR and the intensity of Wip1 immunoblots. (C) miR-16 has a rapid induction in response to DNA damage. U2OS cells were treated with 500 ng/ml NCS and total RNAs were harvested at indicated time points. miR-16 levels were determined by Northern blots (left panel) and quantitative RT-PCR (from 3 sets of samples, right panel). (D) miR-16 counteracts the induction of Wip1 in the DNA damage response. U2OS cells were transfected with control miRNA, miR-16 or antagomir-16. 48 h after transfection, cells were treated with 500 ng/ml NCS and cell lysates were harvested for immunoblotting analysis (left panel). The intensity of Wip1 blots in each sample was quantified by phosphorimager and normalized with the control sample.

Figure 3

MiR-16 suppresses cell proliferation and sensitizes MCF-7 cells to doxorubicin. (A) Growth curves of Wip1+/+ and Wip1−/− MEFs infected with control or miR-16 expressing pseudoviruses. (B) MiR-16 inhibits the expression of Wip1 in MCF-7 cells. MCF-7 cells were transfected with control or miR-16 expression vector DNA. 36 h after transfection, cell lysates were harvested or total RNA was extracted for the analyses of miRNAs (by quantitative PCR, left panel) and proteins (by Western blotting, right panel). (C) MiR-16 sensitizes MCF-7 cells to doxorubicin. MFC-7 cells were transfected with control, Wip1 shRNA, miR-16 expression vector DNA, or miR-16 and Wip1 shRNA expression vector DNA. 48 h after transfection, cells were incubated with increasing doses of doxorubicin as indicated. Cell viability was determined 3 days after incubation.

Figure 4

MiR-16 is downregulated in mammary tumor stem cells. (A) Levels of Wip1 and miR-16 in mouse mammary tumors are not significantly different from those of normal mammary tissues. N1-3: normal mammary gland tissues; T1-3: mammary tumors. Levels of Wip1 mRNA and miR-16 were determined by quantitative RT-PCR. (B) Micrographs of mammospheres originated from mammary tumor stem cells. TC-1, 2: mammary tumor cells; MS-1, 2: mammospheres from the corresponding mammary tumor cells. (C) Expression levels of stem cell markers and differentiation markers in mammospheres and their original tumors. Stem cell markers (Oct-4, KLF-4), differentiation markers (Keratin-14 and Keratin-18), miR-16, and β-actin (as control) were analyzed by semi-quantitative RT-PCR in starting tumor cells and mammospheres. NC: normal tissue cells. (D) miR-16 is upregulated in cells differentiated from mammospheres. Wip1 protein levels were increased in mammospheres, but were downregulated when mammospheres were differentiated to non-stem cells (Rec: recovered cells) (upper panels). In contrast, miR-16 was downregulated in each of the individual mammospheres, but was upregulated in re-differentiated cells. Levels of miR-16 were determined by quantitative RT-PCR (bottom panel).

Figure 5

MiR-16 inhibits the maintenance and proliferation of mammary tumor stem cells. (A) miR-16 and the Wip1 inhibitor reduce the number of mammary tumor stem cells. Cells isolated from mammary tumors in transgenic MMTV-erbB2 mice were infected by control viruses or pseudoviruses expressing miR-21, miR-16, and/or mouse Wip1, and cultured in the mammosphere-forming medium with or without 5 μM of the Wip1 inhibitor CCT007093. 10 days after incubation, mammospheres were observed and their sizes and numbers were determined. (B) Mammosphere formation is inhibited by miR-16, the Wip1 inhibitor, or Wip1 shRNA, and restoration of Wip1 reversed the effects of miR-16 on blocking mammosphere formation in culture. Mammosphere numbers (left panel) and sizes (right panel) were measured under each condition.