Characterizing the expression and function of metastasis suppressor protein Nm23-H1 in triple-negative breast cancer Open Access

Tobin, Rachel Sydney (2015)

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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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