James Perry

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.

Novel Therapies in Glioblastoma

Conventional treatment of glioblastoma has advanced only incrementally in the last 30 years and still yields poor outcomes. The current strategy of surgery, radiation, and chemotherapy has increased median survival to approximately 15 months. With the advent of molecular biology and consequent improved understanding of basic tumor biology, targeted therapies have become cornerstones for cancer treatment. Many pathways (RTKs, PI3K/AKT/mTOR, angiogenesis, etc.) have been identified in GBM as playing major roles in tumorigenesis, treatment resistance, or natural history of disease. Despite the growing understanding of the complex networks regulating GBM tumors, many targeted therapies have fallen short of expectations. In this paper, we will discuss novel therapies and the successes and failures that have occurred. One clear message is that monotherapies yield minor results, likely due to functionally redundant pathways. A better understanding of underlying tumor biology may yield insights into optimal targeting strategies which could improve the overall therapeutic ratio of conventional treatments.

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 LB-102: Cilengitide induces apoptosis and reduces cell viability in breast cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Brain metastasis from breast cancer incidence is increasing as better control of primary disease leads to longer patient survival. Current therapy for brain metastases is whole brain radiotherapy, stereotactic radiosurgery, or surgical resection. Due to the relative impermeability of the blood brain barrier, not may chemotherapeutics can effectively target these metastases in the brain. Cilengitide, an αVβ3 and αVβ5 inhibitor, is a cyclic RGD containing pentapeptide. Cilengitide has been shown to cross the blood brain barrier and accumulate to appreciable levels in brain tissue in a phase 2 trial in glioblastoma. αVβ3 integrins play a role in angiogenesis, but also are expressed on tumor cells and play a role in proliferation, survival, and invasion. The purpose of these experiments was to examine the role of cilengitide in cell proliferation and survival in a panel of breast cancer cell lines. T-47D, MCF-7, MDA-MB-468, and MDA-MB-231 cell lines were examined. Initially, cells were counted after 1hr of treatment with various cilengitide doses. Next, cells were treated for 4 days with the same cilengitide doses and a proliferation assay (MTS) was performed. These assays showed that at short and long timepoints, cilengitide treatment can have a significant effect on cell viability and proliferation. T-47D cells were particularly sensitive to cilengitide at both short and long timepoints, while MDA-MB-468 cells were particularly resistant. The other cell lines showed an intermediate phenotype. In an attempt to understand the fate of cells following treatment, apoptosis was measured by Annexin V/Propidium Iodide staining and flow cytometry after 48hr treatment with various cilengitide doses. This assay showed increased apoptotic and dead cells, which increased in a dose dependent fashion for all cell lines except MDA-MB-468 cells which again showed no response to cilengitide treatment. As was seen previously, T-47D cells were the most sensitive, followed by MCF-7 cells and then MDA-MB-231 cells. Cells were also examined for Integrin β3 expression by western blot, with target identified in all cell lines. In conclusion, we have observed that cilengitide has both immediate and lasting effects on breast cell line viability and proliferation. Not all cell lines responded equally however, and the reason for these differences is not fully understood. Cilengitide target expression level may play a role in this process, and apoptosis appears to be possible mechanism for cilengitide mediated cell death in these cell lines. Cilengitide appears to be a good candidate for personalized therapy, but a more complete understanding of its effects could identify a patient subpopulation that is most likely to respond favorably to cilengitide. Citation Format: James D. Perry, David Peereboom, Arnab Chakravarti, Tim Lautenschlaeger. Cilengitide induces apoptosis and reduces cell viability in breast cell lines. [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 LB-102. doi:10.1158/1538-7445.AM2013-LB-102

Abstract 2229: Galectin-1 reduces the efficacy of PI3K pathway inhibitors in glioblastoma cells

Galectin-1 (Gal-1), is a small ∼14kD carbohydrate binding protein that is known to be significantly upregulated in gliomas, and has also been closely associated with patient survival. PI3K/AKT signaling is important in cancer cells because upregulation of this pathway is associated with increased cell survival and growth. Specifically in glioblastomas there is a signaling alteration in the RTK/PI3K/RAS/PTEN axis in nearly 90% of patients leading to therapeutic resistance and poor prognosis. While Gal-1 has a defined role in activating MAPK signaling, the effect of Gal-1 on PI3K signaling is unclear. The purpose of these experiments was to study the role that Gal-1 has in cellular survival signaling, primarily the PI3K pathway. Previous experiments from our laboratory have demonstrated a significant role for Gal-1 in PI3K signaling and AKT activation. An in vitro PI3K reaction showed that Gal-1 can increase PI3K enzyme activity, even in the presence of a PI3K inhibitor. Gal-1 was also shown to specifically interact with the PI3K enzyme in an in vitro Co-IP assay. Based on these results, we investigated the role of Gal-1 in protecting cells from PI3K pathway inhibition. LN229, U87, and LN18 glioma cell lines were used for these experiments. Cells were treated with multiple different inhibitors for specific proteins in the PI3K signaling pathway (EGFR - Gefitinib, AKT - MK-2206, PI3K - LY294002 and PI-103, mTor - AZD 8055 and Everolimus). Initially, control cells were compared with cells of different Gal-1 status for PI3K pathway activation by western blot. We looked at multiple levels of activation including pAKT, p-mTOR, and p-p70S6K. Following this analysis, proliferation was examined by MTS assay and clonogenic survival was also tested. Our results indicate that in the absence of Gal-1, all of the different inhibitors listed above showed increased signaling inhibition in each of the cell lines tested. In the control cells, higher concentrations of these inhibitors were required to cause similar reduction in pathway activation markers such as pAKT. Based on these results we examined proliferation rates and clonogenic survival for several of the tested inhibitors. In these experiments, we observed that Gal-1 increases baseline survival signaling in cells and leads to an increased resistance to many of the tested inhibitors. In conclusion Gal-1 has demonstrated an ability to protect cancer cells from the effects of multiple PI3K pathway inhibitors (i.e. PI3K, EGFR, and mTOR). Likely, this effect is due to the increased baseline survival signaling seen in Gal-1 expressing cells. While the mechanism for Gal-19s affect on inhibitor activity is still under investigation, this research highlights that Gal-1 status might serve as a predictive biomarker for the effectiveness of many different PI3K pathway inhibitors that are currently showing limited efficacy in clinical trials. 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 2229. doi:1538-7445.AM2012-2229

Abstract 301: Galectin-1 modulates PI3 kinase activity

The purpose of these experiments was to study the role that Galectin-1 (GAL1) has in Phosphoinositide 3-kinase (PI3K) signaling. GAL1 protein levels have been identified to be significantly upregulated in gliomas. GAL1 levels have also been closely associated with patient survival. The PI3K enzyme converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 serves as a membrane anchor leading to the activation of AKT. PI3K/AKT signaling is important in cancer cell biology because induction of this pathway is associated with increased cell survival, cell cycle progression, and growth. A PIP3 Mass ELISA was performed on cell lysates isolated from wild type (WT) and GAL1 knockdown cells with and without Inslulin-like growth factor 1 (IGF1) stimulation. Briefly, cells were stimulated with 5mM IGF1 followed by cell lysis. Lysates were compared by ELISA for PIP3 levels. Following this an in vitro PI3K assay was done. This assay is designed to measure the activity of a purified PI3K enzyme by detecting PIP2 to PIP3 conversion. Enzyme was incubated with Gal1 alone, LY294002 PI3K inhibitor, or with both Gal1 and the inhibitor. Controls included enzyme only, enzyme with inhibitor, no enzyme, and no substrate. In order to investigate whether a physical interaction was occurring between Gal1 and PI3K, Co-IP experiments were performed. These experiments involved isolation of cell lysates from WT LN229 cells and GAL1 knockdown LN229 cells. Following pulldown, Co-IP9s were run on an SDS-PAGE gel, transferred to blots, and probed for GAL1 and p85 α or β. Previous experiments demonstrated a possible role for GAL1 in PI3K signaling and AKT activation. In the PIP3 mass ELISA experiment, WT cells demonstrated elevated cellular PIP3 levels in response to IGF1 stimulation, as expected. GAL1 knockdown cells did not show any increase in PIP3 levels in response to stimulation. We next examined if GAL1 was having a direct effect on PI3K activity. In vitro PI3K reactions showed a specific upregulation of PI3K activity in response to addition of GAL1. In response to 1000-fold changes in GAL1 concentration there was an approximate 2-fold increase in PI3K (p85α/p110α) activity. GAL1 also demonstrated an ability to overcome LY294002 inhibition of the enzyme. Enzyme incubated with both inhibitor and 5uM GAL1 showed 4 times the conversion as compared to enzyme with inhibitor alone. Interaction studies indicate that there is not a physical interaction between GAL1 and either p85 α or β. Future studies characterizing the role of GAL1 in PI3K signaling include repeating the PI3K assay with other PI3K class I isoforms to examine differences. Fluorescence Resonance Energy Transfer (FRET) experiments will also be performed to further test for a physical interaction between PI3K and GAL1. In conclusion GAL1 has demonstrated an ability to effect PI3K activity, and thereby PI3K signaling. However, a physical interaction between GAL1 and PI3K was not observed. 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 301.

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.