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| Pilar Blancafort, Ph.D.
Assistant Professor |  |
Research Interests
The development of invasive and metastatic behavior during cancer progression is a dynamic process thought to require the concerted action of multiple genes and cellular functions, including cytoskeleton remodeling, acquisition of migratory phenotypes, ability to invade the tumor-surrounding tissues, and survival of disseminated tumor cells. The genetic cascades and transcriptional programs cooperatively required for the malignant progression of tumors are largely unknown. We have developed a novel strategy for the identification and regulation of genes that cooperate during tumor progression. This methodology is based on the screening of large libraries of ATFs made of sequence-specific zinc finger (ZF) domains. In one of our projects, our objective is to apply ATF methodology to identify and regulate genes promoting carcinoma cell invasion and metastasis. Our primary goal is to identify molecular markers of cancer cell invasion that could be used as markers or early detectors of disease progression. We hypothesize that 1) some selected ATFs will be able to regulate complex phenotypes, such as cancer cell invasion and metastatic potential and 2) the selected ATFs can be used to identify markers of cancer disease progression.
Our approach to identify markers of cancer progression is based on functional screenings of ATF libraries. We reasoned that delivery of large repertoires of ATFs will generate phenotypic plasticity in a cancer cell population. Furthermore, a functional screen based on activation of cell invasion will yield ATFs promoting invasiveness. Differential expression profiles of ATF-expressing cells will enable the identification of genes whose contribution is necessary for activation of invasive programs. It is expected that some of these markers will be aberrantly expressed in highly metastatic cancer tissue array samples. ATF libraries comprise millions of proteins with unique and predictable DNA-binding specificities with the potential to interact and regulate virtually any gene in the human genome. When delivered into a poorly-invasive, poorly metastatic cell lines, ATF libraries will induce genome-wide experimental perturbations of gene expression resulting in a phenotypically diverse population or cellular library. Some of the ATFs are expected to regulate key cellular targets that cooperate during tumor progression, triggering complex cellular transformations resulting in induction of malignant behavior, such as an enhanced invasion and metastatic potential. Invasion is one of the initial steps in the metastatic process, and involves the degradation of the different components of the extracellular matrix (ECM). Invasive behavior can be studied in vitro by matrigel invasion assays performed in transwells. Selections of ATF activators (where the ZF domains are linked to the VP64 activator domain) will lead to the isolation of ATF proteins activating genetic programs leading to cell invasion. The selection of ATF repressors (where the ZF domains are linked to the KRAB repressor domain) will yield ATFs targeting cellular factors that suppress cell invasion (or potential metastasis suppressor genes).
ATFs activating cell invasion can be used as genetic probes to identify markers of cancer disease progression. Cells expressing a selected ATFs (exhibiting an invasive phenotype) and control cells (not invasive) can be profiled using DNA microarrays. By comparing these expression profiles we can determine the genes differentially regulated by the ATF that are responsible for the induction of cell invasion. The expression profile of ATF-transduced samples will be then compared with existing profiles of highly metastatic cancer samples. Finally, regulation of these markers at protein level in malignant cancer tissues will be investigated using real time PCR, western blot and tissue arrays. In a second project, we also want to use our artificial transcription factors to target specific gene involved in tumor cell invasion and progression. One of our targets is the E48 marker. E48 is a glycosylphosphatidylinositol (GPI) anchored molecule and marker of disseminated tumor cells in cancers of head and neck. E48 is a target of one of our ATFs promoting migration and invasion in a HeLa cell lines. Interestingly, in addition to the squamous carcinomas of head and neck, E48 has also been found up-regulated in a subset of very metastatic breast tumors associated with a very poor prognosis (Charles Perou, UNC, Dept. of Genetics). Our hypothesis is that, like in HeLa cells, high expression of this antigen in squamous carcinomas of head and neck and in breast tumors is associated with enhancement of cell motility and migration. We want to investigate if modulation of E48 activity by ATFs or siRNAs influences invasion and metastasis in breast tumor cells.
In a third project, we are targeting metastatic suppressor as the Maspin gene with ATFs to down regulate metastatic behavior in a mouse model.
E-mail: pilarb@med.unc.edu
Telephone: 919-966-1615
FAX: 919-966-5640
Address: 102A Mary Ellen Jones Building Chapel Hill, NC 27599-7365
