Abstract
Dynamic protein turnover regulated through protein synthesis and degradation ensures cellular growth, proliferation and differentiation and has growing importance in cancer cells. Ndfip1 and Ndfip2 are known activators of the Nedd4 family of E3 ligases and regulate PTEN/Akt and MAP kinase signaling pathways. Autophagy is a major intracellular lysosomal degradation system, serving as a dynamic recycling platform, frequently deregulated in cancer. Acute myeloid leukemia (AML) has a constitutive Akt activation and autophagy suppression. Acute promyelocytic leukemia (APL) has increased protein accumulation and constitutive autophagy activation. Arsenic trioxide (ATO) is an effective treatment for APL and an established autophagy inductor. The crosstalks between ubiquitin proteasome system (UPS) and autophagy are an ongoing work and mostly unknown.
We sought to investigate the expression levels of NDFIP1 and NDFIP2 in AML patients and to evaluate the role of both genes in autophagy. Bone marrow diagnostic samples from 27 patients with AML, 5 patients with APL and 15 healthy donors were analyzed, using qPCR. Functional studies were performed in vitro using the leukemic cell line U937 silenced for NDFIP1 (shNDFIP1) or NDFIP2 (shNDFIP2) through lentiviral particles. After puromycin selection and confirmation of transduction efficiency, cells were cultured upon ATO treatment 1µM or vehicle in the same concentration for 24h in order to induce autophagy. Acridine orange staining was used to quantify the percentage of acid vesicle organelles (AVO) and mitochondrial mass was estimated with Mitotracker Green, analyzed with flow cytometry. The expression of autophagic proteins (PIK3C3, Sqstm1, LC3 I/II) was investigated with western blot.
NDFIP2 expression was significantly decreased in AML (P=0.04) and was increased in APL (P=0.018), compared with healthy donors. NDFIP2 expression also differed between AML and APL (P=0.002). NDFIP1 expression showed no differences among the subgroups analyzed.
shNDFIP1 cells showed increased AVO percentage compared with the control. A slight AVO increase also occurred in shNDFIP2 cells. PIK3C3 expression was increased in shNDFIP1 and shNDFIP2 untreated cells. However, the mitochondrial mass was increased in both silenced cells. Moreover, shNDFIP1 and shNDFIP2 cells do not have modifications in LC3II or Sqstm1 expression.
Interestingly, ATO treatment induced a significant increase of AVO only in control cells (P=0.01), but not in shNDFIP1 or shNDFIP2 cells. Increased AVO in control cells was accompanied by upregulation of PIK3C3 and LC3II and a decrease in Sqstm1. Conversely, ATO induced a downregulation of PIK3C3 and LC3II in silenced cells and upregulation of Sqstm1 in shNDFIP1 cells.
The interplay between autophagy machinery and UPS is important under physiological conditions and has critical role in cancer. Autophagy is an essential adaptive response and modulates leukemic stemness. NDFIP is an endosomal protein with a role in vesicular trafficking. Better knowledge of UPS and autophagy intersections could lead to novel therapeutic targets for AML. Our results suggest that NDFIP is a novel autophagy regulator, as its silencing induces AVO accumulation, but without canonical autophagy activation, since the effector autophagy protein LC3II is decreased. NDFIP could be essential to autophagosome-lysosome fusion and its inhibition would block terminal stages of autophagy
Author notes
Asterisk with author names denotes non-ASH members.
