Characterizing the expression and function of metastasis suppressor protein Nm23-H1 in triple-negative breast cancer Open Access
Tobin, Rachel Sydney (2015)
Abstract
Triple negative breast cancers (TNBCs) are a heterogeneous group
of breast cancers characterized by poor prognoses due to the lack
of three receptors: estrogen receptor (ER), progesterone receptor
(PR), and HER2/neu. As a result, treatments for TNBCs are limited
to conventional chemotherapy, and diagnosis is correlated with
highly metastatic, recurrent, and incurable disease. The metastasis
suppressor protein Nm23-H1 is thought to inhibit metastasis by
controlling cell motility, invasion, cell-cell adhesion, and
cytoskeletal reorganization. We examined the roles of Nm23-H1 in
human TNBC cell lines and its effects on downstream Rho GTPase
signaling, cell migration, and cell invasion. Nm23-H1 protein is
expressed in morphologically distinct TNBC cell lines. Nm23-H1 was
silenced in the epithelial HCC1806 and mesenchymal BT549 cell lines
using transient siRNA techniques to determine the functional roles
of Nm23-H1 in TNBCs. These cells were also treated with megestrol
acetate (MA), a drug that increases Nm23-H1 protein levels, to
determine the effects of increased Nm23-H1 in TNBC cells.
Interestingly, Nm23-H1 appeared to display tissue-specific
functional roles in epithelial versus mesenchymal cells. In the
epithelial HCC1806 cell line, Nm23-H1 acts in its defined role as a
metastasis suppressor. Paradoxically, our data suggests that
Nm23-H1 appears to function as an oncogene in the mesenchymal BT549
cell line. Therefore, Nm23-H1 could serve as a potential
therapeutic target in epithelial but not mesenchymal TNBC
phenotypes, since Nm23-H1 exerts oncogenic effects in mesenchymal
tissue. Our data support the notion that targeting Nm23-H1 in
epithelial TNBC tumors could serve as a novel therapeutic strategy
to reduce the high mortality rates associated with TNBC
metastasis.
Table of Contents
I. Introduction
a. Breast cancer statistics
b. Breast cancer subtypes
c. Triple-negative breast cancer
d. Metastasis dangers and overview of mechanism
e. Epithelial to mesenchymal transition (EMT): an important step in the metastatic cascade
f. Metastasis suppressors and Nm23-H1
g. The Rho GTPases and their oncogenic link
h. Our strategy, hypothesis, and specific aims
II. Nm23-H1 Expression in Triple-Negative Breast Cancer Cell Lines
a. Specific Aim 1
b. Rationale
c. Methods
i. Pathway Studio
ii. Cell lines, Antibodies, and Reagents
iii. Western Blotting
d. Results
III. Chapter 3: Effects of Stable Knockdown of Nm23-H1 on Triple-negative Breast Cancer Cell Migration and Invasion
a. Specific Aim 2
b. Rationale
c. Methods
i. siRNA Silencing
ii. Western Blotting
iii. RT-PCR
iv. Immunocytochemistry
v. Wound-healing Assay
vi. Matrigel Invasion Assay
vii. Spheroid Migration Assay
d. Results
IV. Chapter 4: Effects of Nm23-H1 overexpression on triple-negative breast cancer cell lines
a. Specific Aim 3
b. Rationale
c. Methods
i. Megestrol acetate (MA)
ii. Survival Assay
iii. MA Dose Curve
iv. MA Time Curve
v. Western Blotting
vi. Wound-healing Assay
vii. Spheroid Migration Assay
viii. Matrigel Invasion Assay
ix. GTPase Activity Assay
d. Results
V. Chapter 5: Discussion and Conclusions
VI. References
VII. Figures
a. Figure 1: Expression profile of Nm23-H1 and downstream targets in TNBCs
b. Figure 2: Nm23-H1 silencing in TNBC cell lines
c. Figure 3: ICC analysis of Nm23-H1 siRNA
d. Figure 4: Nm23-H1 silencing effects on TNBC migration and invasion
e. Figure 5: Effects of megestrol acetate (MA) on TNBC cell survival and Nm23-H1 signaling
f. Figure 6: Effects of Nm23-H1 overexpression on TNBC migration and invasion
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