Alexander Huebner

MicroRNA-31 predicts the presence of lymph node metastases and survival in patients with lung adenocarcinoma

Purpose: We conducted genome-wide miRNA-sequencing (miRNA-seq) in primary cancer tissue from patients of lung adenocarcinoma to identify markers for the presence of lymph node metastasis. Experimental Design: Markers for lymph node metastasis identified by sequencing were validated in a separate cohort using quantitative PCR. After additional validation in the The Cancer Genome Atlas (TCGA) dataset, functional characterization studies were conducted in vitro. Results: MiR-31 was upregulated in lung adenocarcinoma tissues from patients with lymph node metastases compared with those without lymph node metastases. We confirmed miR-31 to be upregulated in lymph node-positive patients in a separate patient cohort (P = 0.009, t test), and to be expressed at higher levels in adenocarcinoma tissue than in matched normal adjacent lung tissues (P < 0.0001, paired t test). MiR-31 was then validated as a marker for lymph node metastasis in an external validation cohort of 233 lung adenocarcinoma cases of the TCGA (P = 0.031, t test). In vitro functional assays showed that miR-31 increases cell migration, invasion, and proliferation in an ERK1/2 signaling-dependent manner. Notably, miR-31 was a significant predictor of survival in a multivariate cox regression model even when controlling for cancer staging. Exploratory in silico analysis showed that low expression of miR-31 is associated with excellent survival for T2N0 patients. Conclusions: We applied miRNA-seq to study microRNomes in lung adenocarcinoma tissue samples for the first time and potentially identified a miRNA predicting the presence of lymph node metastasis and survival outcomes in patients of lung adenocarcinoma. Clin Cancer Res; 19(19); 5423–33. ©2013 AACR.

Combined RASSF1A and RASSF2A Promoter Methylation Analysis as Diagnostic Biomarker for Bladder Cancer

Promoter hypermethylation, a widely studied epigenetic event known to influence gene expression levels, has been proposed as a potential biomarker in multiple types of cancer. Clinical diagnostic biomarkers are needed for reliable prediction of bladder cancer recurrence. In this paper, DNA promoter methylation of five C-terminal Ras-association family members (RASSF1A, RASSF2A, RASSF4, RASSF5, and RASSF6) was studied in 64 formalin-fixed paraffin-embedded (FFPE) bladder cancer and normal adjacent tissues using methylation-specific high-resolution melting (MS-HRM) analysis. Results showed that 73% (30/41) of transitional cell carcinoma, 100% (3/3) of squamous cell carcinoma, and 100% (4/4) of small cell carcinoma demonstrated promoter methylation of the RASSF1A or RASSF2A gene, but only 6% (1/16) of normal tissues had promoter methylation of RASSF genes. Testing positive for hypermethylation of RASSF1A or RASSF2A promoter provided 77% sensitivity and 94% specificity for identification of cancer tissues with an area under the curve of 0.854, suggesting that promoter methylation analysis of RASSF1A and RASSF2A genes has potential for use as a recurrence biomarker for bladder cancer patients.

In vitro study of combined cilengitide and radiation treatment in breast cancer cell lines

BackgroundBrain metastasis from breast cancer poses a major clinical challenge. Integrins play a role in regulating adhesion, growth, motility, and survival, and have been shown to be critical for metastatic growth in the brain in preclinical models. Cilengitide, an αvβ3/αvβ5 integrin inhibitor, has previously been studied as an anti-cancer drug in various tumor types. Previous studies have shown additive effects of cilengitide and radiation in lung cancer and glioblastoma cell lines. The ability of cilengitide to enhance the effects of radiation was examined preclinically in the setting of breast cancer to assess its possible efficacy in the setting of brain metastasis from breast cancer.MethodsOur panel of breast cells was composed of four cell lines: T-47D (ER/PR+, Her2-, luminal A), MCF-7 (ER/PR+, Her2-, luminal A), MDA-MB-231 (TNBC, basal B), MDA-MB-468 (TNBC, basal A). The presence of cilengitide targets, β3 and β5 integrin, was first determined. Cell detachment was determined by cell counting, cell proliferation was determined by MTS proliferation assay, and apoptosis was measured by Annexin V staining and flow cytometry. The efficacy of cilengitide treatment alone was analyzed, followed by assessment of combined cilengitide and radiation treatment. Integrin β3 knockdown was performed, followed by cilengitide and radiation treatment to test for incomplete target inhibition by cilengitide, in high β3 expressing cells.ResultsWe observed that all cell lines examined expressed both β3 and β5 integrin and that cilengitide was able to induce cell detachment and reduced proliferation in our panel. Annexin V assays revealed that a portion of these effects was due to cilengitide-induced apoptosis. Combined treatment with cilengitide and radiation served to further reduce proliferation compared to either treatment alone. Following β3 integrin knockdown, radiosensitization in combination with cilengitide was observed in a previously non-responsive cell line (MDA-MB-231). Clonogenic assays suggested little radiosensitization effects of cilengitide.ConclusionsCilengitide appears to enhance radiation response in preclinical models of breast cancer. These data suggest that the combination of radiation therapy and cilengitide may prove to be effective where radiation is utilized for the treatment of gross disease in breast cancer, such as in the setting of brain metastasis.

Abstract 2963: 5-hydroxymethylcytosine alterations at H3K9me3 marked genomic regions serve as potential biomarker for renal cell carcinoma patients

Background: Epigenetic alterations are frequently encountered in many malignancies. Control of DNA methylation is thought to play a critical role in the global epigenetic reprogramming observed during development and carcinogenesis. DNA demethylation is initiated by the transition from 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC patterns are relatively well characterized in many malignancies, not much is known about 5hmC patterns and the potential usefulness as biomarkers. We thus sought out to identify 5hmC alterations in renal cell carcinoma (RCC) and assess their potential usefulness as urine biomarkers. Results: We performed genome-wide 5hmC MethylCap-seq on one pooled RCC tissue sample (n = 3) and the corresponding pooled matched normal kidney tissue (n = 3), as well as on one pooled urine sample obtained from RCC patients (n = 52) and one pooled urine sample obtained from control patients without malignancy (n = 65). Global 5hmC levels were dramatically reduced in RCC tissues compared to matched normal adjacent kidney tissues, and in urine samples compared to tissue samples. Assessing histone marked regions we found 5hmC levels to be highly enriched in H3K9me3 marked repressive genomic regions of RCC tissue compared to normal adjacent tissues. Given the low 5hmC signal in these regions in normal tissues, this difference was also clearly identified comparing urine samples from RCC patients to control patients without RCC. Conclusions: We characterized the 5hmC distribution of RCC and normal human renal tissue as well as of urine samples from RCC patients and control patients without RCC. We identified dramatic genome-wide 5hmC changes to occur during carcinogenesis, which have potential for development as non-invasive urine biomarkers. Citation Format: Wei Meng, Tim Lautenschlaeger, David Frankhouser, Zhenqing Ye, Alexander Huebner, Victor Jin, Pearlly Yan, Arnab Chakravarti. 5-hydroxymethylcytosine alterations at H3K9me3 marked genomic regions serve as potential biomarker for renal cell carcinoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2015-2963

Abstract LB-362: A six-gene classifier stratifies low-grade glioma patients with wild-type IDH

Gliomas are the most common primary brain tumors presenting a heterogeneous group of malignancies. Particularly, there is a significant heterogeneity in outcomes of patients with grade II and grade III gliomas. There has been recent progress in reclassifying glomas by integrating molecular signatures such as IDH mutation status, 1p/19q co-deletion and alternative telomer maintenance to the established histopathological classification scheme. Patients with IDH wild-type (WT) gliomas are found to have the worst prognosis. Currently there is no prognostic or predictive biomarker within this group of patients. To this end, the goal of the current study was to develop a gene classifier for identifying a prognostic/predictive biomarker model for grade II and grade III gliomas with IDH WT, which would have the potential of identifying patient groups with distinct degrees of risk for disease progression, treatment options, and survival outcome. Our approach was to identify a robust list of candidate genes using multiple datasets and create a single score classifier based on the expression of identified genes. We analyzed multiple datasets comprising a total of 756 glioma patients using a meta-analysis approach which identified six prognostic genes. A linear model consisting of these six genes was trained in an institutional cohort by employing RNAseq and qRT-PCR techniques to measure the gene expression and validated in an independent grade II/III glioma set (TCGA LGG). The classifier performed significantly in both the institutional cohort of 85 grade II/III glioma patients (HR, 29.94; 95% CI, 3.96-226.36; p = 0.001) and in the TCGA LGG dataset consisting of 416 glioma patients (HR, 4.03; 95% CI, 1.82-8.92; p = 0.001, for low versus high risk group). Multivariate Cox regression analysis validated the risk classifier independent of other clinical factors. After analyzing patient populations separately, based on IDH mutation status, we discovered that the classifier performed with high significance in the patients with IDH WT both in the institutional cohort and the TCGA LGG dataset (HR, 1.93; 95% CI, 1.24-3.02; p = 0.004 and HR, 1.99; 95% CI, 1.38-2.87; p Citation Format: Alexander Huebner, Oliver Oehlke, Tareq A. Juratli, Wei Meng, Ahmed Ibrahim, Bin Li, Daniel Erny, Pierre Robe, Dietmar Krex, Gabriele Schackert, Marco Prinz, Anca Grosu, Arnab Chakravarti. A six-gene classifier stratifies low-grade glioma patients with wild-type IDH. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-362.

Abstract 2235: GAL-1 function as an alternative PI3K pathway activator depends on PTEN and EGFR status in CHO cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The PI3K signaling pathway, one of the most potent prosurvival pathways, has been associated with adverse outcome in glioma. The frequency of signaling alterations in the RTK/PI3K/RAS/PTEN axis in glioblastoma (GBM) is 88% and leads to therapeutic resistance and poor prognosis. Previous results in our lab have shown that Galectin-1 (GAL-1) gene expression is prognostic in five glioma microarray datasets and in four out of five GBM subpopulations. Our lab also has data suggesting an interaction between GAL-1 and PI3K leading to increased PI3K activity. The purpose of this study was to examine GAL-1s influence on survival in GBM patients in regard to their PI3K status. Therefore an exploratory subgroup analysis of the TCGA GBM dataset was conducted and findings were validated in preliminary cell culture model. The TCGA dataset was normalized using previously described methods. Survival analysis was performed using BRB tools. Survival distributions within the dataset were examined among two predefined subgroups: Patients with both EGFR mRNA expression below the median and PTEN expression above the median formed the “low PI3K” group and all other patients formed the comparison “high PI3K” group. The Kaplan Meier curves generated using graphpad prism were stratified by GAL-1 (median split) expression. Stable constructs of CHO cells transfected with an LGALS1 expression vector and/or with PTEN shRNA were created and transiently transfected with an EGFR expression vector. Western blots and clonogenic survival assay were performed. The group of patients with “low PI3K” activity had better outcome than the patients with “high PI3K”. This result suggests that patients with lack of PI3K activating EGFR over expression or PTEN loss have improved survival outcome. When survival of the “low PI3K” group is stratified for GAL-1 expression we found patients with high levels of GAL-1 to have significantly worse outcomes when compared to patients with low GAL-1 levels within this group. Analyzing survival for the patients with “high PI3K” activity, we did not observe any difference in prognosis when stratifying for GAL-1 expression. These results led to the hypothesis that high GAL-1 results in reduced patient survival through increased PI3K signaling. We hypothesize that the CHO cells without EGFR, reexpressed PTEN and GAL-1 knockdown have the lowest expression of pAKT. Furthermore we hypothesize that these cells are also the most radiosensitive cells in a clonogenic survival assay. These results indicate that GAL-1 could be an important prognostic marker and that over expression of GAL-1 is functionally similar to loss of PTEN or upregulation of EGFR. They also indicate that GAL-1 might be an alternative mediator of PI3K signaling. These results will be validated in a panel of glioma cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2235. doi:1538-7445.AM2012-2235

Abstract 1077: Galectin-1 is implicated in G1/S-checkpoint control following irradiation - possible explanation of Galectin-1 mediated radioresistance

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: The purpose of the conducted study was to examine a possible therapeutic relevance of Galectin 1 (Gal-1), a beta-galactoside binding protein. Gal-1 is overexpressed in high grade gliomas and leads to increased radioresistance in glioma cell culture models. Inhibition of Gal-1 increases radiosensitivity and thus is a potential therapeutic target for radiation sensitization. P53 has been shown to negatively regulate Gal-1, hence radiosensitivity could possibly be cell cycle mediated. Experimental Design: We used flow cytometry based cell cycle analysis to investigate cell cycle distribution of LN 229 glioma cells following irradiation. LN229 control cell lines and shRNA mediated Gal-1 knockdown LN 229 cell line were exposed to 6 Gy of irradiation and cell cycle analysis was performed. Furthermore Western blot analysis was carried out, probing for multiple proteins implicated in cell proliferation. The phosphorylation status of a variety of phosphorylation sites of p53, Chk1/2 and AKT were examined. Additionally p53-Gal-1 double-knockdown cell lines were created and clonogenic survival assays as well as AnnexinV apoptosis assays were conducted to analyze the influence on radiation sensitivity. Results: The results of the flow cytometry based cell cycle analysis show significant differences between the LN 229 control cell line and the Gal-1 knockdown cell line after irradiation. 24 h after irradiation 77 % of the control line cells are in G1 whereas only 60 % of the Gal-1 knockdown cells are in G1. Previous data has identified differences in the phosphorylation status of Akt in control vs. Gal-1 knockdown glioma cell lines. Phosphorylation of Akt is induced after irradiation. Western blot analysis indicates a reduction of phosphorylated Chk1/2 in Gal1 knockdown cell lines after irradiation. The phosphorylation state of the p53 phosphorylation site, correlating with Chk1/2 activity, exhibit anticipated results. P53-Gal-1 double-knockdown cell lines demonstrate significantly reduced clonogenic survival compared to control cell lines. Conclusions: The finding that less Gal-1 knockdown cells are in G1 phase after irradiation and that there is a reduction in phosphorylated Chk1/2 leads to the conclusion that Gal-1 knockdown abrogates G1 arrest through a Chk1/2 mediated pathway. That means in reverse that Gal-1 can mediate cell cycle arrest, thus potentially can explain Gal-1 mediated radioresistance. Further analysis of Chk1/2 independent phosphorylation sites of p53 is going to be conducted. 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 1077.