Novel Therapeutic Strategies for HER2-overexpressing breast cancer Open Access

Gayle, Sylvia Shabaya (2013)

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This dissertation seeks to uncover mechanisms by which resistance to lapatinib develops and proposes novel therapeutic strategies for patients with HER2-overexpressing breast cancer. The standard targeted therapy for HER2-overexpressing breast cancer is the HER2 monoclonal antibody, trastuzumab. Although effective, many patients eventually develop trastuzumab resistance. The dual EGFR/HER2 small molecule tyrosine kinase inhibitor lapatinib is approved for use in trastuzumab-refractory metastatic HER2-positive breast cancer. However, lapatinib resistance is also a problem as most patients with trastuzumab-refractory disease do not benefit from lapatinib for long. Understanding the mechanisms underlying lapatinib resistance may ultimately facilitate development of new therapeutic strategies for HER2-overexpressing breast cancer. Our results indicate reduced sensitivity to lapatinib is associated with an inability of lapatinib to inhibit the two main signaling pathways downstream of HER2, MEK/ERK and PI3K/Akt/mTOR. We genetically and pharmacologically blocked MEK/ERK signaling and evaluated lapatinib response by trypan blue exclusion, anchorage-independent growth assays, flow cytometric cell cycle and apoptosis analysis, and in tumor xenografts. Our results suggest MEK inhibition increases response to lapatinib. In addition, Western blots, immunofluorescence, and immunohistochemistry demonstrated the combination of MEK inhibitor plus lapatinib reduced nuclear expression of the MEK/ERK downstream proto-oncogene FOXM1. Other trypan blue, transfection, anchorage-independent growth assays, and xenograft studies evaluating the role of PI3K/Akt/mTOR in lapatinib response demonstrated transfection of constitutively active Akt reduced lapatinib sensitivity, while kinase-dead Akt increased sensitivity. Knockdown of 4EBP1 also increased lapatinib sensitivity, in contrast to p70S6K knockdown, which did not affect response to lapatinib. Pharmacologic inhibition of mTOR increased lapatinib sensitivity and reduced phosphorylated Akt levels in cells that showed poor response to single-agent lapatinib, including those transfected with hyperactive Akt. The collective findings presented herein provide insight into the mechanisms by which resistance to lapatinib is achieved, and suggest potential therapeutic strategies that could be of benefit to patients with HER2-overexpressing breast cancer.

Table of Contents


Chapter 1. Introduction and Background

1. EGFR Receptor Tyrosine Kinase Family and HER2-Overexpressing Breast Cancer 2

i. HER2 and breast cancer 2

ii.The role of the EGFR family 5

2. Signaling Pathways Downstream of EGFR Family

i. JAK/STAT 10

ii. PLC 11

iii.SGLT 12

iv.PI3K/Akt 13

v. MEK/ERK 17

3. HER2 Targeted Therapies

i. Irreversible pan-HER kinase inhibitors 21

A. Neratinib (HKI-272) 21

B. Canertinib22

C. BIBW-2992 (Tovok) 23

ii. Reversible pan-HER kinase inhibitors 23

A. Lapatinib 23

iii.HER2 Targeted antibodies 25

A. Pertuzumab 25

B. Trastuzumab 26

4. Mechanisms of Resistance to HER2 Targeted Therapies

i. Mechanisms of resistance to trastuzumab 28

A. p27/cdk2 30

B. PTEN deficiency/increased Akt activity 34

C. Src rapid dissociation 36

D. EGFR-overexpression 38

E. IGF-IR 39

ii. Mechanisms of Resistance to lapatinib 48

A. MEK/ERK activity 48

B. Src 48

C. AXL 49

D. PTEN deficiency/increased Akt 49

5. Scope of this dissertation 50

Chapter 2. Materials and Methods

1. General Methods

i. Reagents 53

ii. Bacterial transformations 54

iii.Cell culture 55

iv.Cell cycle analysis 55

v.DNA/siRNA transfection 55

vi.Immunoflourescence 56

vii.Immunohistochemistry 56

viii.Polymerase chain reaction 57

ix.Western blotting 58

x.ELISA 59

2. Biological Assays

i. Cell proliferation assay 59

ii. Trypan blue 60

iii. Anchorage-independent growth 60

3. Xenograft Studies

i. Xenograft mouse model 61

Chapter 3. Pharmacologic Inhibition of mTOR Improves Lapatinib

Sensitivity in HER2-overexpressing Breast Cancer Cells with Primary Trastuzumab Resistance

1. Introduction 63

2. Results

i. Analysis of lapatinib response in HER2-overexpressing breast cancer cell lines with

primary resistance to trastuzumab 68

ii. Constitutively active Akt reduces lapatinib sensitivity, while kinase

dead Akt improves lapatinib sensitivity 72

iii. Knockdown of 4EBP1 but not p70S6K improves lapatinib sensitivity 73

iv. Rapamycin increases lapatinib sensitivity in HER2-overexpressing breast cancer cells that

have primary trastuzumab resistance 79

v. Pharmacologic mTOR inhibitor MK-8669 Increases lapatinib sensitivity of HER2-overexpressing

breast cancer cells 85

3. Discussion 97

Chapter 4. Sustained MEK Signaling Confers Lapatinib Resistance Through FOXM1

1. Introduction 98

2. Results 100

i. Sustained MEK/ERK signaling is associated with poor response to lapatinib 100

ii. Pharmacologic MEK inhibition increases response to lapatinib 103

iii.Knockdown of MEK in combination with lapatinib induces apoptosis of JIMT-1 cells 112

iv.Co-treatment with selumetinib and lapatinib alters FOXM1 expression 116

v. FOXM1 knockdown increases lapatinib sensitivity 119

vi.The combination of selumetinib plus lapatinib suppresses tumor growth of JIMT-1 xenografts 124

3. Discussion 128

Chapter 5. Summary

i. Summary and Conclusions 133

ii. Implications to the field & Clinic 140

iii .Future Directions 141

Chapter 6. References 143

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