Effects of Stable Insulin-like Growth Factor 1 Receptor Knockdown on Triple-Negative Breast Cancer Cells Open Access

Abstract

Next to non-melanomatous skin cancer, breast cancer is the most common cancer diagnosed among American women today. It is estimated that yearly, more than 190,000 women are diagnosed with breast cancer and greater than 40,000 succumb to the disease. Aggressive, metastatic disease is directly responsible for the majority of breast cancer-related deaths. Triple-negative (TN) breast cancers, which lack estrogen receptor, progesterone receptor and HER2/neu overexpression, lead to poorer survival outcomes compared to all other breast cancer patients, partly because of a lack of therapeutic targets. Insulin-like growth factor 1 receptor (IGF-1R) is overexpressed in 50% of primary breast tumors compared with normal tissues and 36% of TN breast cancers express IGF-1R. IGF-1R plays a role in proliferation, apoptosis, adhesion, and invasion, suggesting that breast cancers have enhanced responses to the mitogenic and anti-apoptotic effects of IGF-I. The objective of this project was to determine the significance of differential IGF-1R signaling in the aggressive properties of TN breast tumors. Stable lentiviral IGF-1R knockdown was performed in two morphologically distinct TN breast cancer cell lines, MDA-MB-468 and MDA-MB-231. Knockdown of IGF-1R led to down regulation of AKT signaling as well as lack of IGF-I-induced IGF-1R up-regulation in both IGF-1R (-/-) cell lines compared to empty vector control cell lines. Interestingly, each TN cell line underwent distinct morphological changes in response to IGF-1R silencing. As evidenced by confocal microscopy and Western blot analyses, MDA-MB-468 (epithelial) cells appeared to undergo epithelial to mesenchymal transition (EMT) while MDA-MB-231 (mesenchymal) cells underwent mesenchymal to epithelial transition (MET). Epithelial markers (E-cadherin and β-catenin) and mesenchymal markers (vimentin and fibronectin) were also differentially expressed in each IGF-1R (-/-) cell line. Combinatorial inhibition of IGF-1R, EGFR, and/or mTOR decreased cell survival more efficiently than single inhibition. These results suggest that IGF-1R inhibition, in combination with EGFR and/or mTOR down-regulation may provide clinical benefit in a subset of TN breast cancer patients, particularly those with mesenchymal-like tumor phenotypes.

Table of Contents

Table of Contents

I. Abstract

II. Chapter 1: Introduction

a. Breast Cancer Statistics 1

b. Overview of Breast Cancer Subtypes 1

c. Triple-Negative Breast Cancers 3

d. Basal-like Breast Cancers 3

e. Claudin-low Breast Cancers 5

f. Triple-Negative Breast Cancers-Mechanisms of Malignancy and Role of IGF-1R 6

g. Transcriptional Control of IGF-1R 8

h. Epithelial to Mesenchymal Transition (EMT) 9

i. Tumor Initiating Cells (TICs) 9

j. Oncogene Addiction and Targeted Therapies 10

k. EGFR and mTOR signaling in Triple-Negative Breast Cancer 11

l. Our Strategy: Targeting IGF-1R Signaling in TN Breast Cancer 11

m. Hypothesis and Specific Aims 14

III. Chapter 2: Generation of Triple Negative Cell Lines with A Stably Transfected IGF-1R (-/-) Knockdown Lentivirus

a. Specific Aim 1 15

b. Methods

i. Cell lines, Antibodies, and Reagents 15

ii. Western Blotting 16

iii. RNA preparation and RT-PCR 16

iv. Lentivirus preparation 17

c. Results 18

IV. Chapter 3: Effects of Stable Knockdown of IGF-1R on Triple Negative Breast Cancer Cells

a. Specific Aim 2 20

b. Methods

i. IGF-I stimulation and Western Blots 20

ii. Colony formation assay 20

iii. Immunocytochemistry 20

c. Results 21

V. Chapter 4: Effects of IGF-1R, EGFR, and mTOR Inhibition on Triple-Negative Cell Lines

a. Specific Aim 3 25

b. Methods

i. Combination treatments and Western Blot Analysis 25

ii. Cell survival assay 25

c. Results 25

VI. Chapter 5: Discussion and Conclusions 29

VII. References 38

VIII. Figures 40

Table of Figures

a. Figure 1: Insulin-like growth factor 1 signaling 40

b. Table 1: Primers sequences used for RT-PCR 41

c. Figure 2: Protein and mRNA expression levels 42

d. Figure 3: Stable knockdown of IGF-1R in triple-negative breast cancer cell lines 43

e. Figure 4: IGF-1R knockdown differentially affects colony formation and AKT signaling in triple-negative breast cancer cell lines 44

f. Figure 5: Assessment of EMT in triple-negative breast cancer cells 45

g. Figure 6: Assessment of EMT markers in human triple-negative (TN) breast cancer cells 46

h. Figure 7: Effects of human IGF-1R and EGFR monoclonal antibodies on receptor expression levels in TN breast cancer cell lines 47

i. Figure 8: Combined inhibition effectively reduces IGF1-R signaling and survival in HCC1808 TN breast cancer cells 48

j. Figure 9: Effects of combination inhibitors on IGF-1R and EGFR expression in TN breast cancer cells 49

k. Figure 10: Effects of combined inhibition on TN breast cancer cell survival 50

l. Figure 11: Effects of combined inhibition on TN IGF-1R (-/-) breast cancer cell survival 51

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Health Sciences, Oncology Biology, Cell
Keyword	IGF-1R triple-negative cancer EGFR breast
Committee Chair / Thesis Advisor	Taliaferro-Smith, LaTonia, Emory University School of Medicine
Committee Members	Eisen, Arri, Emory University Orloff, Gregg, Emory University

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