Cancer Letters

Cancer Letters

Volume 225, Issue 1, 8 July 2005, Pages 53-59
Cancer Letters

Upregulated Akt signaling adjacent to gastric cancers: implications for screening and chemoprevention

https://doi.org/10.1016/j.canlet.2004.11.021Get rights and content

Abstract

Most gastric adenocarcinomas arise as a longterm complication of Helicobacter pylori infection of the stomach, but the high prevalence of this infection limits the cost-effectiveness of antibiotic eradication as a cancer prevention strategy. Here we have used phosphorylation-specific antibodies against the Akt kinase consensus sequence to detect downstream substrates of this oncogenic signaling pathway in normal and malignant gastric tissues. In vitro studies confirm that phosphorylation of Akt and its substrates is inducible by epithelial mitogens such as epidermal growth factor (EGF), which is implicated in the pathogenesis of H. pylori gastritis. Control clinical studies confirm far stronger Akt substrate phosphorylation in primary human breast cancers than in matched adjacent normal breast tissues; unexpectedly, however, increased Akt signaling is apparent in both primary stomach cancers and adjacent normal gastric tissues. These findings raise the possibility of a preneoplastic field defect induced in morphologically normal tissues, and suggest that immunoassays of mucosal Akt activity could guide preventive surveillance and/or intervention in patients at risk of gastric cancer. Moreover, since recent reports confirm Akt inhibition by COX-2 inhibitors, these data support the chemopreventive efficacy of such drugs for at-risk individuals.

Introduction

Chronic gastric infection with the acid-resistant bacterium Helicobacter pylori [1] is associated with a six-fold increased risk of stomach cancer [2] which remains the second commonest cause of global cancer mortality [3]. The molecular mechanism whereby H. pylori predisposes to such cancers is not established [4], nor is it yet clear to what extent future cancer risk is reduced by eradication of pre-existing infection [5]. These uncertainties have generated much debate about the cost-effectiveness of detecting and treating H. pylori infections as part of a worldwide cancer prevention strategy [6], [7].

A key cytoprotective peptide in the stomach is the acid-stable ligand epidermal growth factor [8] (EGF; originally isolated from salivary glands as urogastrone [9]). H. pylori infection amplifies EGF signaling within the stomach via multiple mechanisms—including induction of EGF receptor (EGFR) expression in gastric epithelial cells [10], metalloprotease-dependent cleavage of gastric epithelial cell-surface precursor EGFR ligands [11], [12], and stress-induced gastric epithelial cell upregulation of EGF expression via EGFR-dependent positive feedback [13]—all of which are normalized by eradication therapy [14], [15].

Critical to its anti-apoptotic action in the digestive tract, EGF activates the Akt (protein kinase B) signaling pathway [16] which in turn upregulates mucosal angiogenic growth factor activity [17]. The centrality of Akt to gastric physiology and neoplasia was suggested by the original cloning of this oncogene from a gastric adenocarcinoma in which the gene was amplified [18], and by its potent pro-mitogenic action on gastric cancer cells in vitro [19]. Moreover, inhibition of Akt promotes Fas- and/or TRAIL-mediated gastric cancer cell apoptosis [20], [21]—consistent with the frequent association of gastric cancer with deletion or methylation of the PI3K/Akt-inhibitory tumor suppressor gene PTEN [22], [23]—raising the possibility that small-molecule antagonists of this pathway could provide an important therapeutic breakthrough in this currently intractable disease.

We previously developed a technology for assaying kinase signaling in vitro [24] and in vivo [25] using phosphorylation-specific antibodies, and this approach has since been used to develop immunoassays for the functional activity of both the Akt serine/threonine kinase and its downstream substrates [26]. Mass spectrometry has confirmed that the latter phosphoantibody identifies a spectrum of Akt-phosphorylated proteins, including the transcription-regulatory Forkhead (FKHR) proteins [27], the proliferation-regulatory glycogen synthase kinase (GSK-3) isoforms [28], and the translation-regulatory ribosomal S6 protein (RS6) [29]. Here we use these function-specific reagents to assess the relative activity of the Akt signaling pathway in normal human tissues adjacent to either primary breast or gastric adenocarcinomas.

Section snippets

Reagents

EGF and transforming growth factor-beta (TGF-β) were obtained from Sigma Chemical Co. (St Louis, MO), insulin and protease inhibitors from Roche (Mannheim, Germany), phospho-Akt-substrate antibody and anti-phospho-Akt-S473 from Cell Signaling and Transduction Laboratory, respectively, and HRP-conjugated donkey anti-rabbit IgG secondary antibody from Jackson ImmunoResearch (West Grove, PA). Polyvinylidene difluoride (PVDF) membrane was obtained from Millipore (Bedford, MA), and enhanced

Growth factor-dependent stimulation of Akt pathway signaling

For control purposes, the ability of polypeptide growth factors to activate the Akt signaling pathway in vitro using human embryonic kidney (HEK-293) cells was first assessed. As shown in Fig. 1(A), the epithelial growth factors EGF and insulin enhance immunodetection of downstream Akt signaling, whereas a mesenchymal mitogen (transforming growth factor-beta) does not; the time-course of such signaling closely parallels the activation-dependent phosphorylation of the Akt enzyme itself (Fig. 1

Discussion

The central finding of this study is that downstream substrates of Akt signaling are actively phosphorylated—as determined by use of the Akt substrate phosphoantibody (PAS)—in nonmalignant gastric tissues adjacent to primary stomach cancers. This conclusion supports and extends the recent work of Nam et al., who used anti-phospho-Akt (Ser 473) immunohistochemistry to show concomitant Akt activation in normal and malignant gastric epithelium [33]. Interestingly, these authors noted an improved

Acknowledgements

We thank Chow Yin Wong for kind provision of control breast tissues. This work was supported by the National Medical and Research Council of Singapore.

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