title: Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer.
abstract: There 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.
  
  
  
  Phospho-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.
  
  
  
  Immunohistochemistry 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.
  
  
  
  These 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.
authors.affiliation: Department of Medical Oncology VU University Medical Center, Cancer Center Amsterdam, CCA room 1.52, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.|Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, Palermo, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Massihnia
authors.affiliation: Metabolic syndrome Research center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Amir
authors.initials: A
authors.lastname: Avan
authors.affiliation: Cancer Pharmacology Lab, AIRC Start Up Unit, University of Pisa, Pisa, Italy.
authors.forename: Niccola
authors.initials: N
authors.lastname: Funel
authors.affiliation: Department of Medical Oncology VU University Medical Center, Cancer Center Amsterdam, CCA room 1.52, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Mina
authors.initials: M
authors.lastname: Maftouh
authors.affiliation: Department of Medical Oncology VU University Medical Center, Cancer Center Amsterdam, CCA room 1.52, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Anne
authors.initials: A
authors.lastname: van Krieken
authors.affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Granchi
authors.affiliation: Department of Medical Oncology VU University Medical Center, Cancer Center Amsterdam, CCA room 1.52, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Rajiv
authors.initials: R
authors.lastname: Raktoe
authors.affiliation: Department of Surgery, University of Pisa, Pisa, Italy.
authors.forename: Ugo
authors.initials: U
authors.lastname: Boggi
authors.affiliation: Æterna Zentaris GmbH, Frankfurt am Main, Frankfurt, Germany.
authors.forename: Babette
authors.initials: B
authors.lastname: Aicher
authors.affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: Minutolo
authors.affiliation: Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, Palermo, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Russo
authors.affiliation: Cancer Pharmacology Lab, AIRC Start Up Unit, University of Pisa, Pisa, Italy.
authors.forename: Leticia G
authors.initials: LG
authors.lastname: Leon
authors.affiliation: Department of Medical Oncology VU University Medical Center, Cancer Center Amsterdam, CCA room 1.52, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
authors.affiliation: Department of Medical Oncology VU University Medical Center, Cancer Center Amsterdam, CCA room 1.52, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. e.giovannetti@vumc.nl.|Cancer Pharmacology Lab, AIRC Start Up Unit, University of Pisa, Pisa, Italy. e.giovannetti@vumc.nl.
authors.forename: Elisa
authors.initials: E
authors.lastname: Giovannetti
mesh_terms: D000368:Aged; D017209:Apoptosis / Q000187:drug effects; D001706:Biopsy; D021441:Carcinoma, Pancreatic Ductal / Q000175:diagnosis / Q000188:drug therapy* / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D004357:Drug Synergism; D005260:Female; D051272:Glucose Transporter Type 1 / Q000187:drug effects; D006801:Humans; D008297:Male; D008875:Middle Aged; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000188:drug therapy* / Q000473:pathology; D010750:Phosphoproteins / Q000032:analysis / Q000037:antagonists & inhibitors; D011379:Prognosis; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis* / Q000037:antagonists & inhibitors*; D012333:RNA, Messenger / Q000032:analysis; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured; D000093542:Gemcitabine
chemical_list: D051272:Glucose Transporter Type 1; D010750:Phosphoproteins; D012333:RNA, Messenger; C495677:SLC2A1 protein, human; D003841:Deoxycytidine; D051057:Proto-Oncogene Proteins c-akt; D000093542:Gemcitabine
keywords: Akt; Gemcitabine; Pancreatic ductal adenocarcinoma; Synergism