15-Deoxy-Δ^12,14-prostaglandin J₂ activates PI3K-Akt signaling in human breast cancer cells through covalent modification of the tumor suppressor PTEN at cysteine 136

Highlights

• PTEN is inactivated through covalent modification by 15-deoxy-Δ^12,14-prostaglandin J₂ in breast cancer MCF-7 cells.

• 15-Deoxy-Δ^12,14-prostaglandin J₂ directly binds to the cysteine 136 residue of the recombinant PTEN.

• PTEN interaction with 15-deoxy-Δ^12,14-prostaglandin J₂ stimulates Akt phosphorylation.

• 15-Deoxy-Δ^12,14-prostaglandin J₂ stimulates growth of MDA-MB-231 cells when inoculated to the athymic nude mice.

Abstract

15-Deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), one of the terminal products of cyclooxygenase-2-catalized arachidonic acid metabolism, has been shown to stimulate breast cancer cell proliferation and migration through Akt activation, but the underlying mechanisms remain poorly understood. In the present study, we investigated the effects of 15d-PGJ₂ on the activity of PTEN, the inhibitor of the phosphoinositide 3-kinase (PI3K)-Akt axis, in human breast cancer (MCF-7) cells. Since the α,β-unsaturated carbonyl moiety in the cyclopentenone ring of 15d-PGJ₂ is electrophilic, we hypothesized that 15d-PGJ₂-induced Akt phosphorylation might result from the covalent modification and subsequent inactivation of PTEN that has several critical cysteine residues. When treated to MCF-7 cells, 15d-PGJ₂ bound to PTEN, and this was abolished in the presence of the thiol-reducing agent dithiothreitol. A mass spectrometric analysis by using recombinant and endogenous PTEN protein revealed that the cysteine 136 residue (Cys¹³⁶) of PTEN is covalently modified upon treatment with 15d-PGJ₂. Notably, the ability of 15d-PGJ₂ to covalently bind to PTEN as well as to induce Akt phosphorylation was abolished in the cells expressing a mutant form of PTEN in which Cys¹³⁶ was replaced by serine (C136S-PTEN). The present study demonstrates for the first time that electrophilic 15d-PGJ₂ directly binds to cysteine 136 of PTEN and provides new insight into PTEN loss in cancer progression associated with chronic inflammation. These observations suggest that 15d-PGJ₂ can undergo nucleophilic addition to PTEN, presumably at Cys¹³⁶, thereby inactivating this tumor suppressor protein with concomitant Akt activation.

Introduction

Breast cancer is one of the leading causes of cancer-related deaths in women [1, 2]. As in the case of the majority of other malignancies, inflammation is associated with pathogenesis of breast cancer. Cyclooxygenase-2 (COX-2), a key enzyme involved in inflammatory response, is frequently overexpressed in various cancer types including breast cancer [[3], [4], [5], [6]]. Abnormally elevated COX-2 expression or activity causes stimulation of cancer cell proliferation, resistance to cancer cell apoptosis, and enhancement of angiogenesis and invasiveness which account for its oncogenic function [7]. COX-2 catalyzes the conversion of arachidonic acid to a series of prostaglandins (PGs), some of which have been reported to play roles in carcinogenesis [4, 5, 8]. Of note, aberrant induction of COX-2 in breast cancer is correlated with an increased production of 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) [8]. 15d-PGJ₂ has been known to have oncogenic characteristics [9]. 15d-PGJ₂ contains a chemically reactive α,β-unsaturated carbonyl moiety in its cyclopentenone ring. Because of its electrophilic nature, 15d-PGJ₂ can form a covalent adduct with free thiols of cysteine residues present in various proteins via the Michael addition reaction. Proteins which are known to be covalently modified by 15d-PGJ₂ include nuclear factor-kappa B (NF-κB) [10], activator protein-1 (AP-1) [11], H-Ras [12], the tumor suppressor protein p53 [13], eukaryotic initiation factor 4A [14], Rac1 [15], c-Jun [16] and Hu antigen R [17].

Our previous study demonstrated that 15d-PGJ₂ induced COX-2 expression in human breast cancer (MCF-7) cells via the Akt-AP-1 signaling pathway [8]. Akt activation by 15d-PGJ₂ was considered to be responsible for pro-inflammatory and plausible tumor-promoting effects of this cyclopentenone PG [8]. However, the molecular mechanism underlying 15d-PGJ₂-induced Akt activation remains to be clarified. The tumor suppressor, phosphatase and tensin homolog (PTEN) has been known to counteract the function of PI3K and consequently repress the Akt activation via its lipid phosphatase activity [18]. In multiple tumor types, the functional inactivation of PTEN has been observed [[19], [20], [21], [22]]. In line with this notion, the loss of PTEN gene expression is associated with poor outcome in breast cancer [23]. Notably, overexpression of COX-2 inactivates signaling molecules including PTEN [24, 25]. Upon treatment with arachidonic acid, Akt phosphorylation was induced through oxidation and concurrent inactivation of PTEN in COX-2-overexpressing pancreatic cancer (BxPC3) cells [24]. The principal catalytic function of PTEN is to dephosphorylate phosphatidylinositol-3,4,5-triphosphate (PIP₃), which is a potent activator of Akt and 3-phosphoinositide-dependent kinase [26]. The unique PIP₃ lipid phosphatase activity of PTEN makes it one of the most important tumor suppressors. Loss of PTEN function leads to increased accumulation of PIP₃ and sequential reactivation of the PI3K-Akt signaling that stimulates cell growth and survival [27].

Alteration of PTEN functional activity by post-translational modifications has been proposed as a therapeutically attractive strategy [[28], [29], [30], [31]]. Several studies have convinced that the catalytic activity of PTEN is modulated by reactive oxygen species (ROS) and reactive nitrogen species [29, 32, 33]. PTEN contains several conserved cysteine residues that are susceptible to oxidation or nitrosylation. These catalytic cysteine residues might be also potential targets for Michael addition by electrophilic species. Previous studies from this laboratory and others demonstrated the intracellular production of ROS by 15d-PGJ₂. As 15d-PGJ₂ has both pro-oxidant and electrophilic properties, this prompted us to investigate whether 15d-PGJ₂ could induce Akt phosphorylation through oxidation and/or covalent modification and subsequent inactivation of PTEN in human breast cancer cells.