MicroRNAs in pancreatic cancer
MicroRNA-21 from bench to bedside and back: a potential marker of clinical outcome and a target to overcome resistance to gemcitabine in pancreatic cancer
Giovannetti E (1,2), Funel N (3), Erozenci LA (1), Del Chiaro M (3), Leon GL (1), Vasile E (4), Pollina LE (3), Groen A (2) Falcone A (4), Boggi U (3), Campani D (3), Verheul HM (1), Danesi R (2), Peters GJ (1).
(1) VU University Medical Center, Amsterdam, The Netherlands; (2) Dept. Internal Medicine University of Pisa; (3) Divisione di Chirurgia Generale e dei Trapianti dell'Uremico e del Diabetico, Hospital of Pisa; (4) Unit of Medical Oncology-2, Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy
Abstract
MiR-21 was reported to be overexpressed in PDAC and contributed to tumor invasion and resistance to gemcitabine. Further studies are needed to test whether miR-21 could be used to stratify patients for treatment and provide mechanistic insights for new therapeutic targets.
The aim of this study was to evaluate whether miR-21 expression was associated with the overall survival (OS) of PDAC patients treated with gemcitabine, and investigate its role in PDAC invasive behaviour and chemoresistance.
MiR-21 expression was evaluated in pancreatic cells (including 7 PDAC cell lines, 7 primary PDAC cultures, fibroblasts and a normal pancreatic ductal cell line) and tissues (neoplastic specimens from 77 PDAC patients (32 in the palliative and 45 in the adjuvant setting) and normal ductal samples) isolated by laser microdissection. The role of miR-21 on the pharmacological effects of gemcitabine was studied in cells transfected with a specific miR-21 precursor (pre-mir). Modulation of apoptosis, Akt activation and VEGF secretion was analyzed by Annexin V and ELISA assays, respectively. Quantitative PCR evaluated the correlation of miR-21 with invasion-related genes and VEGF. Inhibitors of PI3K and mTOR (LY294002 and rapamycin) were used to test whether modulation of these signalling pathways affected molecular mechanisms activated by miR-21 and gemcitabine.
Patients with high miR-21 expression had a significantly shorter OS both in the palliative and in the adjuvant setting, while a trend toward correlation was found between low expression and clinical benefit. Multivariate analysis in all the patients with adequate follow-up (N=58), confirmed the negative prognostic significance of high miR-21 expression (HR:2.6, 95%CI:1.7-3.4; P:0.001). MiR-21 expression was correlated with gemcitabine resistance in all the PDAC cells, and with the expression in their respective tissues in the primary cultures. Treatment with gemcitabine resulted in a significant increase of miR-21 expression, ranging from 2 to 19-fold, in 13 PDAC cells. Pre-mir transfection significantly decreased apoptosis induction by gemcitabine and up-regulated metalloproteinase-2/-9 and VEGF mRNA expression in 2 selected PDAC cultures (LPc028 and LPc067). In contrast, addition of LY294002 and rapamycin resulted in reduction of phospho-Akt and overcame pre-mir induced resistance to the pro-apoptotic effects of gemcitabine.
In conclusion, miR-21 expression correlated with OS in PDAC gemcitabine-treated patients, and further validation in prospective studies is warranted. Modulation of apoptosis, Akt phosphorylation, and expression of genes involved in invasive behaviour, may contribute to miR-21 role in gemcitabine chemoresistance and provide mechanistic insights for the rational development of new targeted combinations against PDAC.