Abstract
BackgroundThere is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated molecular mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine.MethodsPhospho-Akt expression was evaluated by immunohistochemistry in tissue microarrays (TMAs) with specimens tissue from radically-resected patients (n = 100). Data were analyzed by Fisher and log-rank test. In vitro studies were performed in 14 PDAC cells, including seven primary cultures, characterized for their Akt1 mRNA and phospho-Akt/Akt levels by quantitative-RT-PCR and immunocytochemistry. Growth inhibitory effects of Akt inhibitors and gemcitabine were evaluated by SRB assay, whereas modulation of Akt and phospho-Akt was investigated by Western blotting and ELISA. Cell cycle perturbation, apoptosis-induction, and anti-migratory behaviors were studied by flow cytometry, AnnexinV, membrane potential, and migration assay, while pharmacological interaction with gemcitabine was determined with combination index (CI) method.ResultsImmunohistochemistry of TMAs revealed a correlation between phospho-Akt expression and worse outcome, particularly in patients with the highest phospho-Akt levels, who had significantly shorter overall and progression-free-survival. Similar expression levels were detected in LPC028 primary cells, while LPC006 were characterized by low phospho-Akt. Remarkably, Akt inhibitors reduced cancer cell growth in monolayers and spheroids and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028, while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a reduced expression of ribonucleotide reductase, potentially facilitating gemcitabine cytotoxicity. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. This combination significantly increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells.ConclusionsThese data support the analysis of phospho-Akt expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.
Highlights
There is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance
Patients with low phospho-Akt expression had a median overall survival (OS) of 16.2 months, while patients with a high expression had a median OS of 12.0 months
Glut1 is overexpressed in the cells resistant to Akt inhibition, while its inhibition significantly reduces cell growth and induces apoptosis after gemcitabine/ perifosine treatment Since major oncogenic signaling pathways have been linked to increased glucose metabolism, and previous studies showed that stimulation of Akt1 induces Glut1 mRNA and protein accumulation, [43] we evaluated the expression of this key glucose transporter in the LPC028 and LPC006 cells
Summary
There is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance. Despite extensive preclinical and clinical research, the prognosis of this disease has not significantly improved, with a 5-year survival rate around 7% [1] This dismal outcome can partially be explained by the lack of biomarkers for screening and diagnosis at earlier stages, and by the resistance to most currently available chemotherapy regimens. This resistance has been attributed to both the desmoplastic tumor microenvironment and to the strong inter- and intratumor heterogeneity in terms of complexity of genetic aberrations and the resulting signaling pathway activities, as well as to resistance mechanisms that quickly adapt the tumor to drugs [2]. The phosphatidylinositol-3 kinase (PI3K)/Akt downstream pathway represents an exciting new target for therapeutic intervention, especially because it emerged among the core signaling pathways in PDAC [5, 6], and several known inhibitors are currently in clinical trials (www.clinicaltrials.gov)
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