Sean F. Eddy

NF-κB and Epithelial to Mesenchymal Transition of Cancer

During progression of an in situ to an invasive cancer, epithelial cells lose expression of proteins that promote cell–cell contact, and acquire mesenchymal markers, which promote cell migration and invasion. These events bear extensive similarities to the process of epithelial to mesenchymal transition (EMT), which has been recognized for several decades as critical feature of embryogenesis. The NF‐κB family of transcription factors plays pivotal roles in both promoting and maintaining an invasive phenotype. After briefly describing the NF‐κB family and its role in cancer, in this review we will first describe studies elucidating the functions of NF‐κB in transcription of master regulator genes that repress an epithelial phenotype. In the second half, we discuss the roles of NF‐κB in control of mesenchymal genes critical for promoting and maintaining an invasive phenotype. Overall, NF‐κB is identified as a key target in prevention and in the treatment of invasive carcinomas. J. Cell. Biochem. 104: 733–744, 2008.

Inducible IκB Kinase/IκB Kinase ε Expression Is Induced by CK2 and Promotes Aberrant Nuclear Factor-κB Activation in Breast Cancer Cells

Aberrant activation of nuclear factor-κB (NF-κB) transcription factors has been implicated in the pathogenesis of breast cancer. We previously showed elevated activity of IκB kinase α (IKKα), IKKβ, and protein kinase CK2 in primary human breast cancer specimens and cultured cells. A novel inducible IKK protein termed IKK-i/IKKe has been characterized as a potential NF-κB activator. Here, we provide evidence that implicates IKK-i/IKKe in the pathogenesis of breast cancer. We show IKK-i/IKKe expression in primary human breast cancer specimens and carcinogen-induced mouse mammary tumors. Multiple breast cancer cell lines showed higher levels of IKK-i/IKKe and kinase activity compared with untransformed MCF-10F breast epithelial cells. Interestingly, IKK-i/IKKe expression correlated with CK2α expression in mammary glands and breast tumors derived from MMTV-CK2α transgenic mice. Ectopic CK2 expression in untransformed cells led to increased IKK-i/IKKe mRNA and protein levels. Inhibition of CK2α via the pharmacologic inhibitor apigenin or upon transfection of a CK2 kinase-inactive subunit reduced IKK-i/IKKe levels. Expression of a kinase-inactive IKK-i/IKKe mutant in breast cancer cells reduced NF-κB activity as judged by transfection assays of reporters driven either by NF-κB elements or the promoters of two NF-κB target genes, cyclin D1 and relB. Importantly, the kinase-inactive IKK-i/IKKe mutant reduced the endogenous levels of these genes as well as the ability of breast cancer cells to grow in soft agar or form invasive colonies in Matrigel. Thus, CK2 induces functional IKK-i/IKKe, which is an important mediator of the activation of NF-κB that plays a critical role in the pathogenesis of breast cancer. (Cancer Res 2005; 65(24): 11375-83)

Trastuzumab-Resistant HER2-Driven Breast Cancer Cells Are Sensitive to Epigallocatechin-3 Gallate

Overexpression of the epidermal growth factor receptor family member HER2 is found in approximately 30% of breast cancers and is a target for immunotherapy. Trastuzumab, a humanized monoclonal antibody against HER2, is cytostatic when added alone and highly successful in clinical settings when used in combination with other chemotherapeutic agents. Unfortunately, HER2 tumors in patients develop resistance to trastuzumab or metastasize to the brain, which is inaccessible to antibody therapy. Previously, we showed that the green tea polyphenol epigallocatechin-3 gallate (EGCG) inhibits growth and transformed phenotype of Her-2/neu-driven mouse mammary tumor cells. The different modes of action of EGCG and trastuzumab led us to hypothesize that EGCG will inhibit HER2-driven breast cancer cells resistant to trastuzumab. We studied trastuzumab-resistant BT474 human breast cancer cells, isolated by chronic trastuzumab exposure, and JIMT-1 breast cancer cells, derived from a pleural effusion in a patient who displayed clinical resistance to trastuzumab therapy. EGCG treatment caused a dose-dependent decrease in growth and cellular ATP production, and apoptosis at high concentrations. Akt activity was suppressed by EGCG leading to the induction of FOXO3a and target cyclin-dependent kinase inhibitor p27Kip1 levels. Thus, EGCG in combination with trastuzumab may provide a novel strategy for treatment of HER2-overexpressing breast cancers, given that EGCG can cross the blood-brain barrier.

Cloning and expression of PPARγ and PGC-1α from the hibernating ground squirrel, Spermophilus tridecemlineatus

The peroxisome proliferator-activated receptor (PPAR) family of transcription factors play a key role in lipid metabolism and have been implicated in a number of disease states, most notably of which is obesity. Controlled regulation of lipid metabolism is a key ingredient for successful hibernation. Partial cDNA sequences for one of the PPAR proteins, PPARγ and the PPARγ co-activator (PGC-1α) have been cloned from the hibernating ground squirrel, Spermophilus tridecemlineatus and show differential regulation during hibernation at the mRNA level using relative RT-PCR and at the protein level via immunoblotting in brown adipose tissue (BAT), heart, skeletal muscle and white adipose tissue (WAT). The cDNA sequence for PGC-1α revealed a number of amino acid substitutions and two were worthy of note, one resulting in the loss of a potential protein kinase C (PKC) site, while another resulted in the creation of a PKC site, suggesting that PKC may be important in regulating PGC-1α. RT-PCR revealed a near 2-fold up-regulation of PPARγ in BAT and to a lesser extent (< 1.5-fold) in heart and WAT, while PGC-1α displayed significantly higher levels of expression in skeletal muscle during hibernation (3.1-fold, p < 0.005). The protein levels of PPARγ were significantly increased in BAT and WAT (1.5 and 1.8-fold, respectively) while PGC-1α displayed significant changes in expression in heart (3.5-fold) and skeletal muscle (1.8-fold). Our current findings indicate a role for increased expression of PPARγ and PGC-1α in hibernating animals. (Mol Cell Biochem 269: 175–182, 2005)

RelB NF-κB Represses Estrogen Receptor α Expression via Induction of the Zinc Finger Protein Blimp1

ABSTRACT Aberrant constitutive expression of NF-κB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERα)-negative breast cancers versus ERα-positive ones, due in part to repression of RelB synthesis by ERα signaling. Notably, RelB promoted a more invasive phenotype in ERα-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERα synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERα (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERα-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-κB subunit mediates repression, specifically of ERα, thereby promoting a more migratory phenotype.

Comparative Molecular Physiological Genomics

The use of DNA microarrays has gained wider acceptance as a standard tool for molecular biology studies over the past decade. In particular, biomedical studies embraced this technology as soon as arrays were produced for the common laboratory species. Slower to develop, however, has been the use of microarray screening with non-standard animal models, even though these species present fascinating physiological phenomena for study. The very high cost and huge amount of work involved in developing and producing a DNA array or microarray for a new species is prohibitive for most researchers working in comparative biology. The alternative is to explore the use of heterologous array hybridization, screening for stress-induced gene expression in one species using an array developed for another species. This chapter provides a comprehensive review of the current literature on heterologous DNA array hybridization and explores the factors that must be taken into account when performing heterologous microarray analysis on nonstandard species. Changes in methodology (e.g. hybridization conditions, stringency of washing) to optimize the percent cross reaction, the potential for false positives and false negatives to occur, and techniques for downstream analysis and confirmation of array data are all discussed. Examples of cross-hybridization using human microarrays are discussed using phylogenetically diverse species ranging from ground squirrels to frogs to snails. As with any new technology, the willingness to grasp cross-species analysis has been slow but the future looks bright for heterologous DNA hybridization and microarray analysis now that the initial hurdles have been overcome.

Abstract 2897: Discovery and characterization of driver MAPK and PI3K pathway mutations in tumors and association with drug response in cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The MAPK and PI3K pathways are frequently altered in human cancer and are targeted by dozens of agents in clinical trials. The successful application of these therapies, alone or in combination, may depend on the activation status of both pathways. Next-generation sequencing of cancer exomes provides a unique opportunity to systematically survey pathway alterations in cancer. Using somatic mutation data obtained from The Cancer Genome Atlas, we sought to catalog the members of the MAPK and PI3K pathways with driver mutations, the frequency of occurrence in common cancers and the frequency of co-occurrence. Furthermore, we sought to characterize the association of pathway mutation status with drug response in pre-clinical models. While the MAPK and PI3K pathways were frequently altered, the frequency of single and dual pathway alteration and the altered genes varied substantially across cancer types. The MAPK pathway was most frequently altered in rectal (62%), colon (59%), uterine (31%) and lung adenocarcinoma (45%) but infrequently altered in and breast cancer (4%). KRAS, BRAF and NRAS hotspot mutations were the most common pathway drivers, along with NF1 deleterious mutations in certain cancer types. The PI3K pathway was most frequently altered in uterine (84%), breast (40%) and glioblastoma (41%) but was rarely altered in lung adenocarcinoma (9%). Hotspot mutations in PIK3CA and hotspot and deleterious mutations in PTEN were the most common pathway alterations. In addition, predicted driver mutations occurred in PIK3R1, PIK3R3, MTOR, AKT1 and AKT3. Notably, MAPK and PI3K pathway alterations co-occurred in uterine (30%), colon (17%) and gastric (12%) cancers more so than would be expected by chance (p < 0.02). In contrast, other cancer types favored one pathway almost exclusively and thus had little co-occurrence. For example, breast cancer significantly favored PI3K pathway whereas lung adenocarcinoma favored MAPK pathway. To assess the effect of pathway mutation status on treatment response, we integrated hybrid-capture sequencing data from the Cancer Cell Line Encyclopedia with pharmacological data from over 150 compounds. We found that MAPK and PI3K pathway mutations most significantly associated with sensitivity to MEK and PI3K/AKT/mTOR inhibitors, respectively. Notably though, cell lines with co-occurring MAPK pathway and PIK3CA mutations were insensitive to MEK inhibitors and cell lines with co-occurring PI3K pathway and KRAS mutations were insensitive to PI3K inhibitors. Also, not all pathway mutations conferred equal sensitivity. For example, BRAF mutants were generally sensitive, KRAS mutants were mixed and NF1 mutants were generally insensitive to MEK inhibitors. Taken together, our work highlights the need to consider pathways and co-occurrence in the development of targeted therapies. Citation Format: Mark Tomilo, Paul D. Williams, Emma T. Bowden, Supra R. Gajjala, Santhoshi Bandla, Sean F. Eddy, Seth E. Sadis, Peter J. Wyngaard, Nickolay A. Khazanov, Daniel R. Rhodes. Discovery and characterization of driver MAPK and PI3K pathway mutations in tumors and association with drug response in 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 2897. doi:10.1158/1538-7445.AM2013-2897

Abstract 3665: An EMT gene expression diagnostic predicts resistance to EGFR and MEK-targeted therapies in cell lines and patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The epithelial to mesenchymal transition (EMT) in cancer cells results in the acquisition of metastatic properties and may contribute to chemoresistance. Several studies have shown that transition to a mesenchymal phenotype leads to decreased dependence on EGFR-RAS signaling and insensitivity to EGFR inhibitors. To better understand the importance of EMT as a general predictor of drug response, we defined an EMT gene signature derived from a meta-analysis of differential gene expression signatures representing genes up-regulated following transfection of breast cell lines with various EMT regulators (Taube et al., 2010 Proc Natl Acad Sci USA 107:15449-54). We then determined the expression of the EMT signature across cell line panels and determined whether it predicted sensitivity or resistance to various targeted therapies. Consistent with previous results, expression of EMT signature was significantly associated with resistance to an EGFR inhibitor, lapatinib. Similarly, the EMT signature also predicted resistance to PQIP (IGF1R), GSK1120212 (MEK), GSK690693 (AKT), and perifosine (AKT/PI3K), suggesting that EMT may be a common resistance mechanism to a number of drugs that target growth factor signaling. As more of these targeted agents are entering clinical trials, the ability to characterize the signature may have important implications for drug development. To study the relevance of the EMT signature in clinical tumors, we compared the signature to a collection of tumor co-expression patterns, known as OncoScore modules, which were defined from 40,000+ tumor microarray experiments. Notably, the EMT signature was significantly associated with a major tumor co-expression pattern representing mesenchymal and/or stromal phenotype observed in almost all major solid tumor types. In retrospective microarray scoring analyses of key clinical datasets, the mesenchymal/stromal module predicted resistance to cetuximab. This finding was validated with an independent cohort of colorectal cancer patients treated with cetuximab using the Oncoscore Colon diagnostic. Oncoscore Colon is a qPCR test optimized for formalin-fixed paraffin-embedded tissue that measures the twelve key colon cancer transcriptional modules, including the mesenchymal module. Because the mesenchymal/stromal module monitors a fundamental phenotype of cancer cells important for drug response, this validated qPCR test has broad application to companion diagnostics development and personalized medicine. 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 3665. doi:1538-7445.AM2012-3665

Abstract A19: A qPCR assay, OncoScore Colon, predicts resistance to cetuximab in formalin-fixed, paraffin-embedded colorectal cancer tissue independent of KRAS status

Gene expression modules derived from an unsupervised analysis of 20 independent microarray datasets comprising more than 2,000 colorectal cancer patients were identified. Each module represents a set of highly co-expressed genes related to an important aspect of underlying cancer variability. Modules containing genes related to epithelial and mesenchymal biology associated with sensitivity and resistance to EGFR family targeted inhibitors (gefitinib and lapatinib), respectively. In retrospective analysis of clinical samples, the epithelial-mesenchymal axis associated with cetuximab response in two independent patient cohorts. The first study was a Phase II clinical trial (Khambata-Ford et al., J Clin Oncol, 2007) with accompanying microarray data from pre-treatment metastatic colorectal tumor biopsies. Expression of the modules was determined by normalizing and averaging co-expressed module genes. Patients with a more epithelial and less mesenchymal module expression profile were enriched for cetuximab response. An independent cohort of patients was analyzed using module scores that were generated from a qPCR gene expression module test, OncoScore™ Colon, which quantifies modules by averaging three representative module genes relative to housekeeping genes using formalin-fixed-paraffin-embedded primary tumor samples. In these patients, presence of the mesenchymal module was significantly associated with a decrease in progression free survival. Notably, the status of the mesenchymal module was independent of KRAS mutation status—as KRAS mutations occurred in both mesenchymal module-positive and -negative patients. Further clinical studies are ongoing to continue to support the development of the OncoScore™ Colon assay and to further test the predictive capacity of the module with regards to cetuximab resistance and other MAPK pathway inhibitors. This study demonstrates the value of a gene expression module-based qPCR panel for stratifying colorectal cancer patients for treatment response, and suggests that our approach may have immediate utility for cetuximab treatment response prediction.

Abstract 3149: OncoPredictor: A systematic approach for predicting responsive cancer populations from large scale cell line screening

Investigational new drugs for cancer must demonstrate convincing preclinical efficacy and a compelling strategy to translate preclinical observations to the clinical setting. Personalized medicine approaches are gaining wider acceptance, and large scale cell line databases have demonstrated utility in identifying biomarkers of drug response that can inform clinical development strategies. However, the lack of an integrated platform to translate preclinical biomarker profiles to clinical populations limits the power of this approach. To solve this problem we developed, first, a cell line screening and genomic analysis pipeline that associates drug response across 200+ cell lines with mutation, DNA copy number, and gene expression biomarkers; and second, a parallel database of biomarker frequencies in clinical tumor samples, compiled from all available published genomic data. In the present study, we tested 8 targeted anti-cancer agents and identified cell line biomarkers representing each of the genomic data types – mutation, DNA amplification, and gene over-expression – and then assessed the distribution of these biomarkers across tumor samples. In each case the tumor populations predicted to be responsive by this unsupervised approach were validated by results from clinical trials. We also present an example of biomarker results leading to potential new indications for an approved drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3149. doi:10.1158/1538-7445.AM2011-3149