Date of Award

12-1-2013

Degree Type

Dissertation

University or Center

Clark Atlanta University(CAU)

Degree Name

Ph.D.

Biological Sciences

First Advisor

Valerie A. Odero-Marah, Ph.D.

Abstract

Snail is high in several cancers, correlates with poor clinical prognosis, and associated with increased tumor progression via epithelial-to-mesenchymal transition (EMT). EMT is a latent embryonic program that alters epithelial cells to appear more mesenchymal, regulates embryonic development and wound healing. Snail binds to enhancer box sequences on its target genes like E-cadherin, maspin, and estrogen receptor-alpha (ER-a) to increase EMT. MAP kinase (MAPK/ERK1/2) protein signaling regulates the effects of EMT during tumor progression by regulating cell proliferation, growth, migration, adhesion, invasion, and survival. Recent reports suggest that ERK1 and ERK2 isoforms may function differently although they share similar stimulants and substrates. We investigated the mechanism(s) of Snail-mediated EMT that may be regulated by ERK1/2 in breast cancer cells. Snail expression and phosphorylated ERK (pERK) were higher in breast cancer cells compared to normal breast epithelial cells. Snail siRNA in T47-D and MDA-MB-231 breast cancer cells led to p-ERK relocating from the nucleus to the cytoplasm, and MDA-MB-231 had decreased p-ERK expression. Snail overexpression in MCF-7 Snail cells had increased EMT in vitro and in vivo as compared to MCF-7 Neo (control) cells. MCF-7 Snail had less p-ERK than MCF-7 Neo, which was nuclear-localized. ERK2 isoform activity was also higher in the nucleus of MCF-7 Snail compared to MCF-7 Neo. p-ERK and import protein nucleoporin98 (NUP98) were colocalized at the nuclear membrane in MCF-7 Neo suggesting a shift to the nucleus as cancer progresses. MAPK inhibition decreased cell migration and increased cell adhesion in MCF-7 Snail cells, and also re-induced E-cadherin expression, but decreased adhesion and E-cadherin in MCF-7 Neo. ERK2 isoform regulates EMT because ERK2 siRNA decreased Snail in MCF-7 Snail, but decreased E-cadherin in MCF-7 Neo. MCF-7 cells overexpressing Snail decreased ER-a expression. MCF-7 Snail were less responsive to 4- hydroxytamoxifen (4-OHT) chemotherapy using mitochondrial membrane permeability and cytotoxicity assays. MCF-7 Snail were only sensitive after being treated with 4-OHT at lOuM plus UO126 at lOuM. Collectively, our data suggest that nuclear and cytoplasmic ERK1 isoform activity positively regulates cell adhesion and may have a suppressive role in preventing EMT and breast cancer progression. Conversely, nuclear ERK activity which is predominantly ERK2 isoform activity increases EMT, and promotes resistance to chemotherapy and may promote tumor progression.

Signature Location_Supplemental file.pdf (45 kB)
Notice to Users, Transmittal and Statement of Understanding

Included in

Biology Commons

Share

COinS