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IDENTIFICATION AND CHARACTERIZATION OF THE FRY GENE: A NOVEL TUMOR SUPPRESSOR GENE CORRELATED WITH BREAST CARCINOMA PROGRESSION

by
Jessica Corban Wos Graham
B.S. Rutgers College of Engineering - 2003

Thesis Advisor: Helmut Zarbl, Ph.D.
Graduate Program in Toxicology

EOHSI Building
Conference Room C
Piscataway

Wednesday, November 2, 2011
9:30 A.M.


Abstract

Previously, our laboratory identified Fry, the rat ortholog of the Drosophila melanogaster furry gene, as a mammary carcinoma susceptibility gene using Quantitative Trait Locus mapping in a backcross between the susceptible Fischer 344 and resistant Copenhagen rat strains. In lower eukaryotes, the furry orthologs are implicated in diverse cellular processes including epithelial cell polarity, morphogenesis and proliferation. Consistent with its function as a tumor suppressor gene, we now report that FRY expression is reduced in human breast cancer cell lines and in several clinical breast cancer cohorts.
To elucidate FRY function in mammalian cells, we created two sets of isogenic cell lines: one mammary tumor cell line without (MDA-MB-231) and with ectopic expression of Fry (231wCFry); and one normal mammary epithelial cell line without (MCF-10A) and with FRY-targeting shRNA (10A-shFRY). Our in vitro analyses of these cell lines showed that altered FRY expression dramatically affected cell morphology. Moreover, when xenographed into nude mice, the 231CFry cells demonstrated a dramatic reduction in tumorigenicity (8-fold) and diminished invasiveness into surrounding tissues, while the 10A-shFRY cells remained non-tumorigenic. In-silico analysis of our isogenic cell line pairs suggested that FRY changed gene networks involved in epithelial cell differentiation, adhesion, proliferation, and polarity. These analyses along with protein expression analysis indicated that FRY is affecting the Wnt signaling pathway and epithelial-mesenchymal transition, a process involved in tumor progression.
We next investigated FRY mRNA expression in >3,500 human breast cancers, (Oncomine 3.0 Cancer Profiling Database) and FRY protein expression in several clinical cancer cohorts available as breast tissue microarrays using a FRY anti-peptide antibody (which we designed and validated). Our analyses revealed that decreased FRY mRNA and protein expression was highly correlated with poorly differentiated breast cancer phenotypes such as increasing tumor grade and hormone receptor negative breast cancers, consistent with a role for FRY in tumor progression. Along with being decreased in human mammary tumors and consistent with a role for FRY in the progression of epithelial cell carcinomas, we found that FRY was reduced in human epithelial cell cancers (prostate, ovarian, brain, and lung).


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