Gerald C. Gooden

Genome-wide expression changes induced by HTLV-1 tax: evidence for MLK-3 mixed lineage kinase involvement in Tax-mediated NF-κB activation

The Tax protein of human T-lymphotropic virus type 1 (HTLV-1), an oncoprotein that transactivates viral and cellular genes, plays a key role in HTLV-1 replication and pathogenesis. We used cDNA microarrays to examine Tax-mediated transcriptional changes in the human Jurkat T-cell lines JPX-9 and JPX-M which express Tax and Tax-mutant protein, respectively, under the control of an inducible promoter. Approximately 300 of the over 2000 genes examined were differentially expressed in the presence of Tax. These genes were grouped according to their function and are discussed in the context of existing findings in the literature. There was strong agreement between our results and genes previously reported as being Tax-responsive. Genes that were differentially expressed in the presence of Tax included those related to apoptosis, the cell cycle and DNA repair, signaling factors, immune modulators, cytokines and growth factors, and adhesion molecules. Functionally, we provide evidence that one of these genes, the mixed-lineage kinase MLK-3, is involved in Tax-mediated NF-κB signaling. Our current results provide additional insights into Tax-mediated signaling.

Abstract 4336: Human mesodermal-derived CNS metastasis-associated stromal cells induce a fibrotic response to limit tumor growth

Metastasis to the central nervous system (CNS) remains a major cause of mortality and morbidity in patients with systemic cancer. However, the mechanistic interactions of the neural niche with disseminated tumors cells in CNS metastases (CM) are still poorly understood. To better understand the cross-talk between the neural niche and metastatic tumors, we generated five different patient-derived cell lines (PDCs) originating from surgically resected CM. To assess the genetic and epigenetic characteristics of each PDC, DNA and RNA sequencing, and DNA methylation analysis was performed. Non-tumoral PDCs revealed normal copy number profiles, and retention of germline mutations as seen in patient-matched germline DNA. In contrast, one PDC (CM04) resembled its patient tumor, showing numerous copy number and somatic alterations. RNA-seq and DNA methylation analysis demonstrated that non-tumoral PDCs highly resembled each other, suggestive of a common cell of origin. Additionally, PDCs revealed gene expression signatures associated with cancer associated fibroblasts, epithelial to mesenchymal transition, and mesenchymal stem cells. Further in vivo studies demonstrated that CM04 cells were tumorigenic, whereas non-tumoral PDC (CM08) cells were unable to form tumors in mice. However, CM04:CM08 mixed tumors were significantly smaller than CM04 only tumors and revealed induction of a fibrotic response by immunohistochemistry. These data offer the first evidence that CNS metastasis-associated stromal cells (cMASCs) produce a collagen and fibronectin-rich extracellular matrix constituting a protective host response, which impedes growth of tumor cells. The therapeutic potential of these cells merits further exploration. Citation Format: Christophe Legendre, Gerald C. Gooden, Kyle N. Johnson, Rae Anne Martinez, Mark Bernstein, Jennifer Glen, Jeffrey Kiefer, Aleksander Hinek, Steven A. Toms, Bodour Salhia. Human mesodermal-derived CNS metastasis-associated stromal cells induce a fibrotic response to limit tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4336. doi:10.1158/1538-7445.AM2017-4336

Abstract 3825: Whole genome bisulfite sequencing from plasma of patients with metastatic breast cancer identifies putative biomarkers

Although several improvements in the management of breast cancer(BC) have been made, an estimated 90% of BC related deaths are due to the development of distant metastasis to organs such as lung, bone, liver, and brain. Numerous studies suggest that cell-free (cf)DNA methylation could serve as a useful biomarker for improving clinical management. The objective of our study was to identify novel blood-based biomarkers that can be used to predict BC patients at high risk of distant metastasis. For the first time we performed paired-end whole genome bisulfite sequencing (WGBS) using 15 ng of cf plasma DNA from three pools of samples which included metastatic BC to various organs (M, n = 40), breast cancer free survivors (BCF n = 40) and healthy individuals (H, n = 40). Sequences were aligned using the bismark tool and data analysis was conducted using the R package methylKit to identify base-pair resolution DNA methylation differences between groups. A minimum of 5 reads in each group, delta beta values ≥0.2 and p values Citation Format: Christophe Legendre, Gerald C. Gooden, Kyle N. Johnson, Rae Anne Martinez, Bodour Salhia. Whole genome bisulfite sequencing from plasma of patients with metastatic breast cancer identifies putative biomarkers. [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 3825. doi:10.1158/1538-7445.AM2015-3825

Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis

With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of characterized genes with dbEST identified one library that contained ESTs representing 21 neural crest-expressed genes (library 198). The distribution of the ESTs corresponding to these genes was biased toward being derived from library 198. This is in contrast to the EST distribution profile for a set of control genes, characterized to be more ubiquitously expressed in multiple tissues (P < 1 x 10(-9)). From library 198, a subset of 852 clustered ESTs were selected that have a library distribution profile similar to that of the 21 neural crest-expressed genes. Microarray analysis demonstrated the majority of the neural crest-selected 852 ESTs (Mel1 array) were differentially expressed in melanoma cell lines compared with a non-neural crest kidney epithelial cell line (P < 1 x 10(-8)). This was not observed with an array of 1,238 ESTs that was selected without library origin bias (P = 0.204). This study presents an approach for selecting tissue-appropriate cDNAs that can be used to examine the expression profiles of developmental processes and diseases.

Determining the quantitative accuracy of cDNA microarrays

Gene expression profiles for large sets of genes can be generated simultaneously with cDNA microarrays. The quantitative accuracy of this assay requires that the immobilized probes be present in abundant, non-limiting amounts relative to the amount of labelled target. When this is true, the rates of hybridization for the cDNA targets assayed will follow pseudo first-order kinetics and be limited only by the relative concentrations of the probes themselves, providing linear increases of signal relative to input target. We have examined how the levels of PCR product in the printing solution and the amounts of labelled cDNA target in the hybridization change net detectability and ratio stability in our assays, and will present statistical evaluations of their effect on reliability.

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.