Current Research

In recent years, the Ahn lab has focused on the following areas: the detection of low-frequency mutations on a genome-wide level using a novel deep sequencing technology termed Duplex Sequencing (DS); and the regulation of cell function by microenvironments such as extracellular matrix (ECM)-biomimetic nanotopography. We have applied these cutting-edge experimental approaches to study genomic changes and migratory phenotypes of breast cancer and glioblastoma. The ultimate goals of the Ahn lab’s current and future research projects are to identify biomarkers for the early detection of therapy resistance and tumor recurrence using cutting-edge deep sequencing and ECM-biomimetics technologies, and to develop a novel strategy for preclinical screening of anti-migratory chemotherapeutic agents.

 

Ongoing Projects

 

Mutagenesis and migration in glioblastoma cells

Glioblastoma, the most aggressive brain cancer, invariably reoccurs after surgery and rapidly develops resistance to radiation therapy and chemotherapy. The invasive nature of glioblastoma is a major cause of therapeutic failure. Furthermore, the study of glioblastoma invasion is particularly challenging due to the lack of good experimental models that recapitulate the tumor microenvironment. For genetic characterization of tumors, most studies have investigated clonal (high-frequency) mutations and have not analyzed sub-clonal (low-frequency) mutations due to the high error rates (10-1 to 10-3) of DNA sequencing methods. Therefore, little is known about genetic variations and heterogeneities that can determine the evolution of invasive and therapy-resistant subpopulations of glioblastoma cells. To recapitulate in vivo 3D tumor microenvironments, we have applied nanotopographically defined extracellular matrix (ECM)-mimetic culture platforms. To accurately detect subclonal mutations as well as clonal mutations, we have employed Duplex Sequencing (10-8 to 5x10-8), which is >10,000-fold more accurate than other high-throughput sequencing methods. 

      The overall goal of this project is to characterize genetic variations underlying the invasiveness and radiation resistance of glioblastoma cells in biomimetic culture. Aims of this project are to: 1) examine the migration properties of patient-derived orthotopic glioblastoma xenograft (GBM/PDX) and radiation-resistant GBM/PDX (r-GBM/PDX) cells in a nanotopographical ECM-mimetic culture under hypoxia. Cell migration property will be monitored as an indicator of invasion via a high-throughput live-cell microscopy; and 2) determine how the mutation load or mutation types that change during the development of radiation resistance correlate with the migration properties of GBM/PDX and r-GBM/PDX cells. We will identify clonally expanding subclonal mutations and de novo mutations that arise during radiation resistance using Duplex Sequencing. This project has implications in the preclinical screening of anti-migratory chemotherapeutic agents, the molecular classification of glioblastoma, and the identification of biomarkers for the early detection of therapy resistance and tumor recurrence.

 

Genome-wide profiling of mutations in glioblastoma (GBM)

To maximize the scope and impact of this relatively large project, we collaborate with multiple laboratories (Drs. Lawrence A. Loeb, Ray Monnat, Tom Walsh, and Suleyman Gulsuner at the University of Washington and Dr. Charles Cobbs at Swedish Neuroscience Institute).

 

GBM recurrence. Our overall objective is to elucidate the role of subclonal mutations in glioblastoma and to identify and characterize subclonal mutations and mutation profiles that are associated with therapy resistance and glioblastoma recurrence. The focus of this project is to determine if the presence of large numbers of subclonal mutations in glioblastoma underlies the more malignant phenotype of glioblastoma. We have obtained multiple GBM biospecimens (primary and recurrent tumors from matching patients plus peripheral blood mononuclear cells). We have sequenced primary and recurrent GBMs for clonal mutations of the whole exome using whole exome sequencing and for subclonal mutations of the selected genes, which have been reported to be frequently mutated in GBM, using Duplex Sequencing. We are in process of analyzing the sequencing data by determining frequencies, types, and sequence contexts of mutations, and distances between adjacent mutations and correlating our data with the catalogue of somatic mutations in cancer (COSMIC) mutation signatures and with patients’ clinical features.

 

Mutational heterogeneities within individual GBM patients. We also investigate genome-wide mutation profiles within individual glioblastoma patients using whole exome sequencing and Duplex Sequencing. We have obtained several different sections from geographically different locations within each glioblastoma patient. Studies are underway to identify and characterize mutational variations within individual glioblastoma patients using our bioinformatics analysis tools for the sequencing data.

 

Genomic variations during transformation of breast stem cells into tumorigenic cells

Genome sequencing studies have investigated high-frequency (clonal) mutations and have not explored low-frequency (subclonal and rare) mutations due to high error rates of conventional sequencing methods, which obscure true mutations. Thus, little is known about these low-frequency mutational variations that arise during progression of normal cells to tumorigenic cells. We perform a comprehensive mutation analysis of the whole exome and the selected genes in a human breast stem cell-derived carcinogenesis model. To investigate changes in clonal mutations of nuclear DNA that occur as human normal breast epithelial cells are transformed to tumorigenic cells, the whole exome and the entire regions of 81 genes, which are associated with breast and ovarian cancers, were examined using whole exome sequencing and a targeted sequencing in collaboration with Dr. Tom Walsh (UW Medical Genetics). To accurately determine rare and subclonal mutations of the selected genes, we have applied Duplex Sequencing. We are in process of analyzing the sequencing data by determining frequencies, types, and sequence contexts of mutations, and distances between adjacent mutations and comparing and correlating our data with the cancer genome atlas (TCGA) data and with the catalogue of somatic mutations in cancer (COSMIC) mutation signatures.

 

Other emerging projects

 

• Defining the transcriptomic basis for glioblastoma cell invasiveness

• Changes of mitochondrial subclonal mutations and mitochondrial protein expressions in breast normal cell senescence

 

Recently Completed Projects

 

• Regulation of breast cancer stem cell migration by nanotopographical cues

• Mutational characterization of the whole mitochondrial genome in immortalization of different breast epithelial cell types

• Detection of low-frequency mutations and identification of heat-induced artifactual mutations

• Ultra-accurate genome-wide analysis of mitochondrial DNA mutations during breast stem cells into tumorigenic cells

• Detection of ultra-rare mitochondrial mutations in breast stem cells

• Regulation of sphingosine-induced apoptosis by MYCN in PAX3-FOXO1-positive alveolar rhabdomyosarcoma

• The expression changes of DNA-repair related genes during breast stem cell tumorigenesis