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WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.

ABSTRACT: Members of the WD-repeat protein interacting with phosphoinositides (WIPI) family are phosphatidylinositol 3-phosphate (PI3P) effectors that are essential for the formation of autophagosomes. Autophagosomes, unique double-membraned organelles, are characteristic for autophagy, a bulk degradation mechanism with cytoprotective and homeostatic function. Both, WIPI-1 and WIPI-2 are aberrantly expressed in several solid tumors, linking these genes to carcinogenesis. We now found that the expression of WIPI-1 was significantly reduced in a large cohort of 98 primary acute myeloid leukemia (AML) patient samples (complex karyotypes; t(8;21); t(15,17); inv(16)). In contrast, the expression of WIPI-2 was only reduced in acute promyelocytic leukemia (APL), a distinct subtype of AML (t(15,17)). As AML cells are blocked in their differentiation, we tested if the expression levels of WIPI-1 and WIPI-2 increase during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of APL. According to the higher WIPI-1 expression in granulocytes compared with immature blast cells, WIPI-1 but not WIPI-2 expression was significantly induced during neutrophil differentiation of NB4 APL cells. Interestingly, the induction of WIPI-1 expression was dependent on the transcription factor PU.1, a master regulator of myelopoiesis, supporting our notion that WIPI-1 expression is reduced in AML patients lacking proper PU-1 activity. Further, knocking down WIPI-1 in NB4 cells markedly attenuated the autophagic flux and significantly reduced neutrophil differentiation. This result was also achieved by knocking down WIPI-2, suggesting that both WIPI-1 and WIPI-2 are functionally required and not redundant in mediating the PI3P signal at the onset of autophagy in NB4 cells. In line with these data, downregulation of PI3KC3 (hVPS34), which generates PI3P upstream of WIPIs, also inhibited neutrophil differentiation. In conclusion, we demonstrate that both WIPI-1 and WIPI-2 are required for the PI3P-dependent autophagic activity during neutrophil differentiation, and that PU.1-dependent WIPI-1 expression is significantly repressed in primary AML patient samples and that the induction of autophagic flux is associated with neutrophil differentiation of APL cells.

SUBMITTER: Brigger D

PROVIDER: S-EPMC4123064 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.

Brigger D D Proikas-Cezanne T T Tschan M P MP

Cell death & disease 20140703

Members of the WD-repeat protein interacting with phosphoinositides (WIPI) family are phosphatidylinositol 3-phosphate (PI3P) effectors that are essential for the formation of autophagosomes. Autophagosomes, unique double-membraned organelles, are characteristic for autophagy, a bulk degradation mechanism with cytoprotective and homeostatic function. Both, WIPI-1 and WIPI-2 are aberrantly expressed in several solid tumors, linking these genes to carcinogenesis. We now found that the expression o ...[more]

PMID: 24991767

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Project description:All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance is a major problem influencing the efficacy of ATRA. Identification of mechanisms of ATRA resistance are urgenly needed. In the present study, we found that expression of C/EBP?, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBP? were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Moreover, C/EBP? expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cells, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBP? suppression in APL cells. We restored C/EBP? P42 and P30 by lentivirus vectors in NB4-R1 cells, respectively, and found C/EBP? P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence of myeloid differentiation. Further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBP? P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensitivity of NB4-R1 cells was enhanced by restoration of C/EBP? P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBP? expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, the present study demonstrated that suppression of C/EBP? P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensitivity of APL cells. Restoring C/EBP? P42 is an attractive approach for differentiation therapy in ATRA resistant APL.

Dataset Information

WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.

Publications

WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.

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