Phosphoinositide-3-kinase inhibition elevates ferritin level resulting depletion of labile iron pool and blocking of glioma cell proliferation

Highlights

• Gliomas contain high endogenous PI3K activity that controls cell proliferation.

• Inhibition of PI3K regulates ferritin at transcript and translation level.

• PI3K inhibition results decreased labile iron pool and affects cell proliferation.

• Silencing of ferritin-H reverses labile iron pool and cell proliferation.

• This is found only in glioma cells but not in primary astrocytes.

Abstract

Background

Elevated endogenous phosphoinositide-3-kinase (PI3K) activity is critical for cell proliferation in gliomas. Iron availability is one of the essential factors for cell growth and proliferation. However, any relation between PI3K and cellular iron homeostasis has not been understood so far.

Methods

Glioma cells and human primary astrocytes were treated with class I PI3K inhibitors to examine regulation of iron homeostasis components. Regulation of ferritin was detected at mRNA and translational level. Labile iron pool (LIP) and cell proliferation were examined in glioma cells and human primary astrocytes.

Results

Blocking of PI3K activity elevated ferritin level by 6–10 folds in glioma cells by augmenting mRNA expression of ferritin subunits and also by influencing ferritin translation. IRE-IRP interaction was affected due to conversion of IRP1 to cytosolic aconitase that was influenced by increased iron-sulfur scaffold protein iron-sulfur cluster assembly enzyme (ISCU) level. Elevated ferritin sequestered LIP to affect cell proliferation that was reversed in silencing ferritin by siRNAs of ferritin-H and ISCU. Human primary astrocyte with little PI3K activity did not show any change in ferritin level, LIP and cell proliferation by PI3K inhibitors.

Conclusions

PI3K inhibition promotes ferritin synthesis by dual mechanism resulting sequestration of iron to limit its availability for cell proliferation in glioma cells but not in primary astrocytes.

General Significance: This observation establishes a relation between PI3K signalling and iron homeostasis in glioma cells. It also implies that activated PI3K controls ferritin expression to ensure availability of adequate iron required for cell proliferation.

Introduction

The phosphoinositide-3-kinase family of genes encodes lipid and protein kinases to regulate multiple cellular processes including cell survival, cell proliferation, cell cycle progression, angiogenesis, invasion and metastasis [[1], [2], [3]]. PI3Ks are divided into three classes based on their structure and substrate specificity [4]. The class I PI3Ks phosphorylate and activate Akt (also known as PKB) to participate in cell signalling pathways involved in cell proliferation and several other important cellular mechanisms [5,6]. Class IA PI3Ks are diverse in mammals as they have three catalytic p110 isoforms (p110α, p110β, and p110δ; each encoded by a separate gene) and seven regulatory adaptor proteins. The p110γ is the sole class IB PI3K and differs from class IA enzymes in the N-terminus end (lacking p85 binding site) with a p101 or p84 regulatory subunit [7]. Class I PI3Ks are major focus of research as they are coupled to extracellular stimuli and involved in a wide range of cellular processes [[8], [9], [10]]. PI3K phosphorylates the 3’-OH position of the inositol ring of inositol phospholipids to generate phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) [11]. Phosphorylation and activation of Akt (PKB), a serine/threonine kinase, the key mediator of signalling downstream of PI3 kinase, is principally dependent on the production of PIP3 [12].

Glioblastoma multiforme (GBM) is the most common primary tumour of the central nervous system in adults [13] with limited patient survival and is considered to be among the most lethal cancers [14]. The PI3K pathway is frequently over-activated in GBM due to gain-of-function mutations in the structural gene for p110α (PIK3CA) and by loss of phosphatase and tensin homolog (PTEN), a lipid phosphatase and negative regulator of PI3K signalling [15]. It has been reported that PI3K-Akt signalling is elevated in about 88% of all glioblastomas [15,16]. Similar deregulation of the PI3-kinase signalling pathway through mutation of PIK3CA (p110α) and loss of PTEN has also been detected frequently in glioma cell lines [[17], [18], [19]]. It is well established that inhibition of PI3K signalling affects proliferation in GBM primary cells [20,21].

Role of iron in cell proliferation and cell cycle progression has been well documented in cancer cells including gliomas [22,23]. Cellular iron acquisition and retention of excess iron contribute to tumour initiation and growth [23,24]. Cells acquire iron mainly through transferrin receptor-1 (TfR1) to increase labile iron pool (LIP). However, in astrocytes iron is taken up mainly by divalent metal transporter-1 (DMT1) [25]. From LIP, iron is stored within ferritin [26] and utilized in iron containing proteins and enzymes required for cellular homeostasis. Ferritin molecule contains up to 4000 iron atoms in its mineral core. Cytoplasmic ferritin has two subunits of H– (Ft-H) and L-type (Ft-L) and their ratio varies in different cells. Ft-H is widely considered of having ferroxidase activity that helps in mineralization of iron. Recently, poly-r(C) binding proteins (PCBPs) are identified as iron chaperons for loading iron into ferritin [27]. In response to altered cellular iron pool, both Ft-H and Ft-L are regulated at the translational level [26]. They contain a single iron responsive element (IRE) in their 5’untranslated regions (UTR) [24,26]. In iron depleted cells, iron regulatory proteins (IRP1 and IRP2) are activated to bind IRE and subsequently decrease ferritin translation. IRP1 converts to cytosolic aconitase by gaining iron-sulfur cluster while IRP2 is degraded by proteasomal pathway in higher cellular iron level to promote ferritin translation [28]. The iron-sulfur scaffold protein iron-sulfur cluster assembly enzyme (ISCU) is involved in converting IRP1 to cytosolic aconitase [29]. There are a few reports of ferritin regulation at transcriptional level but its regulation at translational level has been studied extensively [23]. The unique iron exporter ferroportin releases excess iron from cellular iron pool [30]. Intracellular iron pool is maintained for different cellular functions including cell proliferation by coordinated regulation of components involved in iron uptake, iron release and iron storage [23,28].

An iron acquisition phenotype has been reported in number of cancers including glioblastomas [31,32]. Considering the role of iron and PI3K activity in proliferation of cancer cells [22,23], a relation between elevated PI3K and iron homeostasis is expected but has not been addressed so far in any cancer cells including gliomas. Here we report that blocking of PI3K activity by various inhibitors of class I PI3K increases Ft-H and Ft-L synthesis by increasing transcripts of ferritin subunits as well as at translational level by affecting IRE-IRP interaction. Resultant higher ferritin level sequesters iron to decrease LIP resulting inhibition of cell proliferation. These effects are observed only in glioma cells but not in primary astrocytes. This study identifies iron sequestration in ferritin as a novel mechanism by which PI3K inhibition results in affecting cell proliferation in gliomas.