The goal of my research program at the Sloan-Kettering Institute over the past three decades plus has been the understanding of the natural history of genomic instability in cancer cells and its implication to clinical behavior of tumors on the one hand, and to normal cellular developmental pathways on the other. Over this time period, the tools to identify, measure, and analyze the mechanisms of genomic instability have evolved remarkably; part of the effort of my program has been to integrate the evolving methodology into an ongoing inquiry into instability of tumor genomes. We have pursued this approach to many issues in tumor biology in reference to the two predominant tumor systems that we have been interested in, B-cell non-Hodgkin's lymphomas (NHL) and male germ cell tumors (GCT). The choice of these tumors as model systems for our studies was based on the fact that both arise in differentiating stem cells providing unusual opportunities to address neoplastic transformation in conjunction with normal and abnormal differentiation of stem cells. More recently, we have become interested in clear cell renal cell carcinoma (RCC) as a model for genetic analysis of tumor metastasis and a treatment-resistant cancer.
Current research of the laboratory falls in two areas: (1) genomic instability, gene discovery, and biological and clinical markers and (2) regulation of pluripotency and lineage differentiation in human cells.
Genomic Instability, Gene Discovery, and Biological and Clinical Markers
Issues currently addressed in this area include progression of follicular lymphoma to higher histologic grades and transformation to diffuse large B cell lymphoma and development of gene expression and DNA copy number change signatures to predict clinical outcome of GCT and RCC, and gene discovery. The methods currently used in these studies include gene expression profiling, genome copy number change analysis by array comparative genomic hybridization (a-CGH), analysis of micro RNAs, and methylation changes using high density microarrays.
Regulation of Pluripotency and Lineage Differentiation in Human Cells
Our interest in issues of pluripotency and lineage regulation stem from our long-standing interest in adult male GCTs. These tumors arise in pluripotent germ cells and display embryonal-like differentiation. They represent a unique convergence between stem cell biology and tumor biology and may be considered as the prototypical cancer stem cells. The histologic GCT subset embryonal carcinoma (EC), exhibits many of the same properties as embryonic stem cells (ES), including expression of the core stem cell transcription factors (TFs) POU5F1 (OCT3/4), NANOG, and SOX2, as well as of stem-cell-specific surface markers such as TRA-1-81, SSEA3, and SSEA4. However, whether or not EC and ES cells utilize the same TF networks and pathways to arrive at the same lineage end point is unknown. In vivo, pluripotent EC cells differentiate into tumors of embryonic (teratoma) and extra-embryonic lineages (choriocarcinoma and yolk sac tumor). Cell lines derived from EC tumors also retain pluripotency and, in common with ES cells, differentiate into multiple lineages in response to morphogens such as bone morphogenetic protein 2 (BMP2) and all-trans-retinoic acid (ATRA) in vitro.
By generating gene expression profiles (GEP) of a large panel of GCTs representing all lineages, we developed a robust histologic classification based on genes that regulate lineage development and identified up-regulation of the stemness genes GDF3, NANOG, and STELLAR (located within a 200kb gene cluster) and a number of transformation and cell-growth-associated genes (CCND2, RASK), all mapped to 12p. We hypothesized that the gain/amplification of 12p, a unique and universal feature of these tumors, is selected to retain pluripotency and self-renewal characteristic of stem cells as well as gain a malignant growth potential, by transformed germ cells.
The availability of GEP data allowed to us to apply a Systems Biology approach to further investigate issues of pluripotency and lineage differentiation in malignant and normal pluripotent cells in collaboration with the group of Andrea Califano at Columbia University. We applied two reverse-engineering algorithms called ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks) and MRA (Master Regulator Analysis) to the GEP data set, to dissect the transcriptional networks and key regulators that control GCT pluripotency and lineage differentiation and developed a GCT cell-specific TF network that we termed GCTNetTF (Figs .1,2). Such an approach is not feasible by using existing ES cell GEP data sets because their sample size is too small and they lack the heterogeneity required for ARACNe to accurately infer TF targets. Within the GCTNetTF, TFs that are known to maintain pluripotency or implement lineage-specific differentiation appear tightly clustered within linear pathways suggesting that these networks could be helpful in identifying additional key regulators of these processes. Indeed, MRA analysis of a separate GEP data set from time course experiments in the differentiation of the EC cell line 27X-1 to endodermal lineage in response to BMP2, based on the GCTNetTF, have identified a panel of Master Regulator Transcription Factors (MRTFs) that appear to be essential for the maintenance of pluripotency and/or induction of lineage specification. The goal of our research now is to Identify and biologically characterize MRTFs and their targets that regulate pluripotency and lineage differentiation of ES and EC cells in response to various morphogens. Biological validation of the role of these TFs and their targets is achieved by gene knock-down and gene up-regulation experiments using lentiviral vectors and lineage modulation in in vitro experiments. Biochemical validation of target interactions is achieved by methods such as chromatin immunoprecipitation (ChiP).
These studies represent a unique and novel systems biology-based approach to identification of TFs and their targets that are essential for maintenance of pluripotency and induction of lineage differentiation. We expect that they will clarify the TF networks utilized by malignant and non-malignant pluripotent cells and serve as a model to identify TFs and their targets required for differentiation of human pluripotent cells into any inducible lineage, with obvious implications to regenerative medicine.