A polymorphism in the promoter is associated with EZH2 expression but not with outcome in advanced pancreatic cancer patients

Aim: EZH2 expression is a prognostic marker in radically resected pancreatic ductal adenocarcinoma (PDAC) patients. Here we investigated its role in locally advanced/metastatic patients, as well as candidate polymorphisms. Materials & methods: EZH2 expression and polymorphisms were evaluated by quantitative reverse transcription PCR in 32 laser microdissected tumors, while polymorphisms were also studied in blood samples from two additional cohorts treated with gemcitabine monotherapy (n = 93) or polychemotherapeutic regimens (n = 247). Results: EZH2 expression correlated with survival and with the rs6958683 polymorphism in the first cohort of patients, but this polymorphism was not associated with survival in our larger cohorts. Conclusion: EZH2 is a prognostic factor for locally advanced/metastatic PDACs, while candidate polymorphisms cannot predict clinical outcome. Other factors involved in EZH2 regulation, such as miR-101, should be investigated in accessible samples in order to improve the clinical management of advanced PDAC

Abstract 3082: Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2

Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5-year survival rate of merely 8%. The tumor microenvironment of PDAC is one of the factors contributing to drug resistance. More specifically, the hypoxic tumor core and the metabolic switch to aerobic glycolysis (the Warburg effect), contribute to the lack of drug response. Therefore, we investigated the effect of several novel lactate dehydrogenase (LDH-A) inhibitors (N-Hydroxyindole-based LDH-A inhibitors, NHI-1 and NHI-2, and the glucose conjugate NHI-Glc-2) in PDAC cells in vitro and in vivo, in combination with the standard drug gemcitabine. For this purpose we used our primary PDAC cancer cell cultures, tested growth inhibition with the SRB chemosensitivity assay, used 3D cultures and established an in vivo orthotopic bioluminescent model. Additionally, LDH-A enzyme activity inhibition by NHI-Glc-2 was assessed by spectrophotometry. LDH-A is overexpressed in PDAC and its expression is correlated with the prognosis of metastatic PDAC. The glucose transporter 1 (GLUT-1) is also overexpressed in PDAC, which would enable an increased uptake of NHI-Glc-2 by the tumor cells. LDH-A mRNA expression and enzyme activity were about 2-fold higher under hypoxic conditions. NHI-1, NHI-2 and NHI-Glc-2 were 4-15-fold more effective under hypoxic conditions compared to normoxia, but gemcitabine was 10-20-fold less active under hypoxia. NHI-1 showed a synergistic effect with gemcitabine in hypoxic PANC-1 and LPC006 cells (combination index 0.14 ± 0.06 and 0.29 ± 0.53, respectively). NHI-Glc-2 inhibited PDAC cell growth in micromolar range under hypoxic conditions and also showed a synergistic effect with gemcitabine. In a 3D spheroid culture (with a hypoxic core), NHI-Glc-2 disrupted the spheroid integrity. Moreover, in an orthotopic PDAC model NHI-Glc-2 showed a more pronounced inhibition (almost complete) of tumor growth compared to gemcitabine. NHI-Glc-2 also showed a favorable pharmacokinetics with a peak plasma concentration of 26 µM at 4 hr, which is higher than the IC50. In conclusion, LDH-A is a viable target in PDAC, and novel LDH-A inhibitors offer an innovative therapeutic tool. Remarkably, the LDH-A inhibitors NHI-1 and NHI-2 increased the effect of gemcitabine under hypoxic conditions, while the glucose conjugated NHI-Glc-2 showed an improved uptake possibly because of the increased GLUT-1 expression, leading to a pronounced in vivo effect

Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer

Abstract Background 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. Methods Phospho-Akt expression was evaluated by immunohistochemistry in tissue microarrays (TMAs) with specimens tissue from radically-resected patients ( n \u2009=\u2009100). 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. Results 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 ..

Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer

Background: 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. Methods: 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. Results: 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. Conclusions: 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

A polymorphism in the promoter is associated with EZH2 expression but not with outcome in advanced pancreatic cancer patients

Aim: EZH2 expression is a prognostic marker in radically resected pancreatic ductal adenocarcinoma (PDAC) patients. Here we investigated its role in locally advanced/metastatic patients, as well as candidate polymorphisms. Materials & methods: EZH2 expression and polymorphisms were evaluated by quantitative reverse transcription PCR in 32 laser microdissected tumors, while polymorphisms were also studied in blood samples from two additional cohorts treated with gemcitabine monotherapy (n = 93) or polychemotherapeutic regimens (n = 247). Results: EZH2 expression correlated with survival and with the rs6958683 polymorphism in the first cohort of patients, but this polymorphism was not associated with survival in our larger cohorts. Conclusion: EZH2 is a prognostic factor for locally advanced/metastatic PDACs, while candidate polymorphisms cannot predict clinical outcome. Other factors involved in EZH2 regulation, such as miR-101, should be investigated in accessible samples in order to improve the clinical management of advanced PDAC

<i>AKT1</i> and <i>SELP</i> Polymorphisms Predict the Risk of Developing Cachexia in Pancreatic Cancer Patients

<div><p>Pancreatic ductal adenocarcinoma (PDAC) patients have the highest risk of developing cachexia, which is a direct cause of reduced quality of life and shorter survival. Novel biomarkers to identify patients at risk of cachexia are needed and might have a substantial impact on clinical management. Here we investigated the prognostic value and association of <i>SELP-rs6136</i>, <i>IL6-rs1800796</i> and <i>AKT1-rs1130233</i> polymorphisms with cachexia in PDAC. Genotyping was performed in DNA from blood samples of a test and validation cohorts of 151 and 152 chemo-naive locally-advanced/metastatic PDAC patients, respectively. The association of <i>SELP-rs6136</i>, <i>IL6-rs1800796</i> and <i>AKT1-rs1130233</i> polymorphisms with cachexia as well as the correlation between cachexia and the candidate polymorphisms and overall survival were analyzed. Akt expression and phosphorylation in muscle biopsies were evaluated by specific ELISA assays. <i>SELP-rs6136-AA</i> and <i>AKT1-rs1130233-AA/GA</i> genotypes were associated with increased risk of developing cachexia in both cohorts (<i>SELP: p</i> = 0.011 and <i>p</i> = 0.045; <i>AKT1: p</i> = 0.004 and <i>p</i> = 0.019 for the first and second cohorts, respectively), while patients carrying <i>AKT1-rs1130233-GG</i> survived significantly longer (<i>p</i> = 0.002 and <i>p</i> = 0.004 for the first and second cohorts, respectively). In the multivariate analysis <i>AKT1-rs1130233-AA/GA</i> genotypes were significant predictors for shorter survival, with an increased risk of death of 1.7 (<i>p</i> = 0.002) and 1.6 (<i>p</i> = 0.004), in the first and second cohorts, respectively. This might be explained by the reduced phosphorylation of Akt1 in muscle biopsies from patients harboring <i>AKT1-rs1130233-AA/GA</i> (<i>p</i> = 0.003), favoring apoptosis induction. In conclusion, <i>SELP</i> and <i>AKT1</i> polymorphisms may play a role in the risk of cachexia and death in PDAC patients, and should be further evaluated in larger prospective studies.</p></div