PML nuclear bodies contribute to the basal expression of the mTOR inhibitor DDIT4

Jayme Salsman,Ellen Parker,Christopher Eskiw,Livia E Anthes,Daniel Léger,Kara L Koskowich,Stephen B Montgomery,Dudley Chung,Kevin S Smith,Graham Dellaire,Kimberly R Kukurba,Alex Stathakis,Laura D Frost,Daniel Gaston,Ian C Chute,Stephen M Lewis,Sara Lahsaee

doi:10.1038/srep45038

Abstract

The promyelocytic leukemia (PML) protein is an essential component of PML nuclear bodies (PML NBs) frequently lost in cancer. PML NBs coordinate chromosomal regions via modification of nuclear proteins that in turn may regulate genes in the vicinity of these bodies. However, few PML NB-associated genes have been identified. PML and PML NBs can also regulate mTOR and cell fate decisions in response to cellular stresses. We now demonstrate that PML depletion in U2OS cells or TERT-immortalized normal human diploid fibroblasts results in decreased expression of the mTOR inhibitor DDIT4 (REDD1). DNA and RNA immuno-FISH reveal that PML NBs are closely associated with actively transcribed DDIT4 loci, implicating these bodies in regulation of basal DDIT4 expression. Although PML silencing did reduce the sensitivity of U2OS cells to metabolic stress induced by metformin, PML loss did not inhibit the upregulation of DDIT4 in response to metformin, hypoxia-like (CoCl2) or genotoxic stress. Analysis of publicly available cancer data also revealed a significant correlation between PML and DDIT4 expression in several cancer types (e.g. lung, breast, prostate). Thus, these findings uncover a novel mechanism by which PML loss may contribute to mTOR activation and cancer progression via dysregulation of basal DDIT4 gene expression.

Highlights

The promyelocytic leukemia (PML) protein is a tumor suppressor and reduced PML expression is associated with several cancers[1]
In both cells lines, silencing of PML was associated with decreased levels of damage inducible transcript 4 (DDIT4) protein (Fig. 1A), with U2OS cells showing a greater decrease than normal human diploid fibroblasts (NHDF) cells (57% vs 47%, Fig. 1B)
Since PML is not known to act directly as a transcription factors (TFs), these results indicate that the significant changes in endogenous DDIT4 transcription triggered by PML loss may be occurring at the level of chromatin and/or subnuclear chromosome organization, where PML nuclear bodies (PML NBs) may play a role in organizing chromosomal domains[14]

Summary

Introduction

The promyelocytic leukemia (PML) protein is a tumor suppressor and reduced PML expression is associated with several cancers (e.g. breast, prostate and lung)[1]. Many of the more than 150 nuclear proteins known to associate with PML are implicated in gene regulation including transcription factors (TFs) (e.g. p53, STATs, SP1), TF regulators (e.g. pRb, CK1, HIPK2, SENPs) and chromatin modifiers (e.g. HDACs, Daxx)[4,7,8]. DDIT4 positively regulates the mTOR inhibiting tuberous sclerosis complex (TCS1/2)[22], and is upregulated in response to various cellular stresses, including DNA damage[23], hypoxia[24,25] and energy stress[26]. We demonstrate here for the first time that PML loss leads to reduced basal DDIT4 expression, which can, in turn alter DDIT4-dependent effects on mTOR activation following various cellular stresses. We demonstrate that the chromosomal locus containing the DDIT4 gene is directly associated with PML NBs, and that DDIT4 and PML expression levels are significantly correlated in several solid tumours including breast, lung and ovarian cancers

Methods

Results

Conclusion