Normal Pancreatic Ductal Cell Line Research Articles

Abstract 2787: Modulation of autophagy and oncogenic phenotypes through CYB5A-TRAF6 signaling influence prognosis of resected and metastatic pancreatic cancer

Abstract High-resolution array comparative genomic hybridization in a cohort of 44 radically resected pancreatic cancer patients revealed a correlation between shorter survival and loss of the 18q22.3cytoband. The present study investigated the genes encoded by this cytoband and provided mechanistic insights on their role in the suppression of oncogenic properties. We studied mRNA and protein expression of FBXO15, C18orf55, CYB5A, CPGL and CPGL-B in frozen laser-microdissected tissues from radically resected (N=48) and biopsies from metastatic patients (N=50). Low CYB5A expression correlated with 18q22.3 deletion and shorter survival, both in resected and metastatic patients (i.e. 6.5 vs. 12.7 months, P=0.004). Association with outcome was validated in a tissue-microarray of a second cohort of radically resected patients (N=100): patients with low expression levels of CYB5A had significantly shorter OS, and multivariate analysis confirmed CYB5A prognostic relevance (increased risk of death=2.0, P=0.02). The role of CYB5A was evaluated in 11 pancreatic cancer cell lines, 5 primary cultures, and a normal pancreatic ductal cell line through retrovirus-mediated up-regulation and siRNA. With these analyses we characterized a novel function of CYB5A, autophagy induction, concomitant with reduced proliferation and migration/invasion of pancreatic cancer cells. Marked accumulation of autophagic vacuoles was detected by electron microscopy, while immunofluorescence revealed CYB5A modulation of LC-3. Activation of pro-autophagic pathways was corroborated by gene and kinase arrays showing a significant up-regulation of several ATG-genes, accompanied by down-regulation of BCL-2 and MAPK14. Additionally, inhibition of multiple components of EGFR, Akt and Src signalling favored cancer cell death while preventing potentially deleterious cross-talk between key oncogenic players. Network analysis suggested CYB5A interaction with TRAF6, which was confirmed by TRAF6 down-regulation after CYB5A reconstitution (-69% in SU.86.86-CYB5A+, P=0.005). CYB5A silencing had opposite effects, restoring TRAF6 expression and wound-healing. In vivo studies were performed in genetically and histopathologically characterized patient-derived CYB5A+ orthotopic models (N=6 mice/group), monitored by Firefly and Gaussia-luciferase bioluminescence, MRI and high-frequency-ultrasound. CYB5A induced autophagy, as demonstrated by immunohistochemical analyses of p62 down-regulation, and ATG7/ATG16L2 overexpression, while inhibiting tumor growth/metastasis and increasing survival (57 vs. 44 days, P=0.03). These results define CYB5A as a novel prognostic factor, exerting its tumor-suppressor function via autophagy-induction and TRAF6 modulation, which holds a potential as a novel therapeutic approach in the clinical management of pancreatic cancer. Citation Format: Elisa Giovannetti, Qiuyan Wang, Niccola Funel, Amir Avan, Tonny Lagerweij, Viola Caretti, Ugo Boggi, Yisong Wang, Sara Caponi, Arjan van der Velde, Enrico Vasile, Henk M. Verheul, Thomas Wurdinger, Giuseppe Giaccone. Modulation of autophagy and oncogenic phenotypes through CYB5A-TRAF6 signaling influence prognosis of resected and metastatic pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2787. doi:10.1158/1538-7445.AM2014-2787

Abstract 1143: Unraveling the role of CYB5A in pancreatic ductal adenocarcinoma (PDAC): correlation with clinical outcome and functional characterization in the modulation of autophagy and oncogenic phenotypes.

Abstract Loss of 18q22.3 was reported to be a prognostic factor in PDAC. This study aimed at evaluating whether the genes of this cytoband were associated with PDAC outcome and provides mechanistic insights on their role in the suppression of oncogenic properties. The expression of FBXO15, C18orf55, CYB5A, CPGL and CPGL-B was evaluated in cells (11 PDAC cell lines, 5 primary cultures, and a normal pancreatic ductal cell line) and in 48 stage IIB PDAC specimens, isolated by laser-microdissection after surgical resection. Low CYB5A expression correlated with 18q22.3 deletion and shorter survival (16.3 vs. 29.5 months, P=0.01). Association with outcome was validated in a tissue-microarray of a second cohort radically resected patients (N=100): patients with CYB5A protein expression levels below median value had significantly shorter OS, and multivariate analysis confirmed CYB5A prognostic relevance (increased risk of death, 2.0, P=0.02). CYB5A expression in primary cultures correlated with expression in their respective tissues, and CYB5A retrovirus-mediated up-regulation of both PDAC-2 primary cells and Su86.86 cells suppressed proliferation and migration/invasion, while enhancing apoptosis and autophagy induction. Marked accumulation of autophagic vacuoles was detected by electron microscopy, while immunofluorescence revealed CYB5A colocalization with LC-3. Activation of pro-autophagic pathways was corroborated by PCR arrays showing a significant up-regulation of several ATG-genes, accompanied by down-regulation of BCL-2 and MAPK14. The phosphorylation status of MAPK14 was also inhibited, together with phospho-EGFR and the pro-invasive kinases phospho-Src/STAT6, as revealed by 144-kinase peptide substrate arrays. Network analysis suggested that this down-regulation was caused by CYB5A interaction with the NF-κB activator TRAF6, whose expression was significantly reduced after reconstitution of CYB5A in a genetic and histopathologic characterized patient-derived orthotopic mouse model. Furthermore, CYB5A upregulation increased survival, while decreasing primary tumor dimension and metastatic spread, as longitudinally monitored with Firefly- and Gaussia-luciferase. We thus identified CYB5A as a novel prognostic factor that modulates autophagy and oncogenic phenotypes, holding a potential as a novel therapeutic approach in the clinical management of PDAC patients. Citation Format: Elisa Giovannetti, Qiuyan Wang, Amir Avan, Niccola Funel, Elena Glavani, Tonny Lagerweij, Davide Chiasserini, Jih-Hsiang Lee, Viola Caretti, Matilde Masini, Ugo Boggi, Yisong Wang, Enrico Vasile, Godefridus J. Peters, Thomas Wurdinger, Giuseppe Giaccone. Unraveling the role of CYB5A in pancreatic ductal adenocarcinoma (PDAC): correlation with clinical outcome and functional characterization in the modulation of autophagy and oncogenic phenotypes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1143. doi:10.1158/1538-7445.AM2013-1143

MicroRNA-21 in Pancreatic Cancer: Correlation with Clinical Outcome and Pharmacologic Aspects Underlying Its Role in the Modulation of Gemcitabine Activity

MicroRNA-21 (miR-21) was reported to be overexpressed and contributes to invasion and gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC). 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 to provide mechanistic insights for new therapeutic targets. miR-21 expression was evaluated in cells (including 7 PDAC cell lines, 7 primary cultures, fibroblasts, and a normal pancreatic ductal cell line) and tissues (neoplastic specimens from 81 PDAC patients and normal ductal samples) isolated by laser microdissection. The role of miR-21 on the pharmacologic effects of gemcitabine was studied with a specific miR-21 precursor (pre-miR-21). Patients with high miR-21 expression had a significantly shorter OS both in the metastatic and in the adjuvant setting. Multivariate analysis confirmed the prognostic significance of miR-21. miR-21 expression in primary cultures correlated with expression in their respective tissues and with gemcitabine resistance. Pre-miR-21 transfection significantly decreased antiproliferative effects and apoptosis induction by gemcitabine, whereas matrix metalloproteinase (MMP)-2/MMP-9 and vascular endothelial growth factor expression were upregulated. Addition of inhibitors of phosphoinositide 3-kinase and mammalian target of rapamycin resulted in decrease of phospho-Akt and prevented pre-miR-21-induced resistance to the proapoptotic effects of gemcitabine. miR-21 expression correlated with outcome in PDAC patients treated with gemcitabine. Modulation of apoptosis, Akt phosphorylation, and expression of genes involved in invasive behavior may contribute to the role of miR-21 in gemcitabine chemoresistance and to the rational development of new targeted combinations.

Abstract 4819: MicroRNA-21 (miR-21) in pancreatic ductal adenocarcinoma (PDAC): Correlation with clinical outcome and pharmacological aspects underlying its role in the modulation of gemcitabine activity

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 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 metastatic/inoperable and 45 in 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 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 adjuvant and inoperable/metastatic setting, while a trend toward correlation was found between low expression and clinical benefit. Multivariate analysis in all 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 the expression in their respective tissues in the primary cultures and with gemcitabine resistance in all the PDAC cells. Treatment with gemcitabine resulted in a significant increase of miR-21 expression, ranging from 2 to 19-fold, in 13 PDAC cell cultures. Pre-mir transfection significantly decreased apoptosis induction by gemcitabine and up-regulated metalloproteinase-2/−9 and VEGF mRNA expression in 2 selected PDAC cultures. In contrast, addition of LY294002 and rapamycin resulted in reduction of phospho-Akt and prevented pre-mir induced resistance to gemcitabine. In conclusion, miR-21 expression was correlated with OS in PDAC gemcitabine-treated patients. In addition, miR-21 expression is associated with reduced apoptosis, increased Akt phosphorylation and expression of genes involved in invasive behaviour resulting in gemcitabine chemoresistance, which was prevented by Akt inactivation. Our findings indicate that inactivation of PI3K/mTOR pathway may increase gemcitabine sensitivity in PDAC and warrants clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4819.

The Stem Cell Factor–c-kit System and Mast Cells in Human Pancreatic Cancer

Stem cell factor (SCF) and its receptor c-kit take part in the regulation of developmental processes of mast cells, hematopoietic stem cells, and melanocytes, as well as in the growth control of human malignancies. To explore the possible role of the SCF–c-kit system and of mast cells in pancreatic cancer, the concomitant expression and distribution of the two molecules were examined in 17 normal and 26 cancerous human pancreatic tissues and in 6 cultured pancreatic cancer cell lines. Mast cell distribution was also evaluated in the same tissue samples. In addition, the effects of SCF and of the c-kit tyrosine-kinase inhibitor STI571 on the growth of the cancer cell lines and of the normal pancreatic ductal cell line TAKA-1 were assessed. SCF immunoreactivity was absent in acinar, ductal, and islet cells of the normal pancreas and faint in pancreatic cancer tissues and cell lines. In contrast, c-kit was clearly present in some normal and hyperplastic ducts of the normal pancreas, in the cancer cells of 73% of the tumor samples, and in all the cell lines tested. Mast cells, identified by tryptase and chymase immunostaining on consecutive tissue sections, showed immunoreactivity for SCF and c-kit in both normal and cancerous specimens and their number was significantly increased (p = 0.03) in pancreatic cancer compared with the normal pancreas. SCF showed a dose-dependent growth inhibitory effect on TAKA-1 cells (p < 0.001), whereas pancreatic cancer cells were resistant to the SCF-induced growth inhibition. Nonetheless, the growth of TAKA-1 cells and pancreatic cancer cells was inhibited by the c-kit tyrosine kinase inhibitor STI571. In conclusion, the SCF–c-kit system, possibly with the contribution of mast cells, may have a growth-regulating role in the normal pancreas, which is altered during malignant transformation.