EZH2 and STING as therapeutic targets in immunologically cold breast and lung tumors Open Access

Abstract

Despite therapeutic advances, cancer remains a leading cause of morbidity and mortality worldwide. In early tumor development, cancer cells often coopt immune cells to promote angiogenesis and avoid immune attacks. They mutate to resist therapy, evade anti-tumor immunity, and metastasize. Cancer cells’ interactions with tumor-infiltrating neutrophils (TINs) have historically received less attention than anti-tumor CD8+ T cells. This dissertation examines the influence of TINs in two treatment-resistant cancers, triple negative breast cancer (TNBC) and lung adenocarcinoma (LUAD). At the molecular level, we highlight the role of enhancer of zeste homolog 2 (EZH2), a pro-tumor methyltransferase, and one of its immunological targets, the stimulator of interferon genes (STING). First, we used CRISPR to develop EZH2 gene knockout (KO) and overexpression (OE) clones of the 4T1 TNBC mouse model and examined downstream effects on invasive and replicative capacities of cancer cells in vitro and in vivo. We observed significant reductions in tumor growth and lung metastasis, as well as dramatic reductions in the ratios of TINs to both CD4+ and CD8+ T cells when EZH2 was knocked out. Results suggest that EZH2 is not only important for tumor growth and metastasis, but that its expression in tumor cells can alter the infiltration and relative role of pro- and anti-tumor immune subsets in the microenvironment. Second, we tested whether chemical inhibition of EZH2 or activation of STING altered LUAD/TIN interplay. We utilized a novel in vitro co-culture model based on human neutrophil migration through LUAD cells grown at an air-liquid interface. Treatment with MSA-2 (STING agonist) resulted in high induction of IL-29 (interferon l3) secretion in EZH2-high LUAD/TIN co-cultures. In contrast, treatment with EPZ6438 and MS1943 (EZH2 inhibitors) modestly influenced mediator secretion without any change in IL-29. Results suggest that STING activation and interferon signaling can be restored by drugs in EZH2-high LUAD/TIN. Collectively, our findings provide insights into the role of TINs, offering novel perspectives on their interactions with cancer cells and other immune cells in the tumor microenvironment. They also advance our knowledge on EZH2 and STING pathway modulation and inform the development of more effective immunotherapeutic approaches for treatment-resistant cancer. 

Table of Contents

Table of contents

 

Chapter 1: Introduction. 1

Overview of the cancer development process. 2

Anti-tumor immunity. 5

Cold tumors. 6

Hot tumors. 8

Tumor-infiltrating neutrophils (TINs) 10

Detailed account of tumor-TIN crosstalk at different cancer stages. 13

Local invasion. 13

Intravasation. 15

Transport in circulation. 16

Arrest in microvessels and extravasation. 17

Formation of micro- / macro-metastases 18

Role of the EZH2 pathway in anti-tumor immunity. 19

Research focus of this thesis manuscript 23

References 25

 

Chapter 2: Research manuscript #1. 35

Abstract 36

Introduction. 37

Materials and methods. 39

Results. 45

Discussion. 49

Acknowledgments. 54

Figures. 55

References 64

 

Chapter 3: Research manuscript #2. 70

Abstract 71

Introduction. 72

Materials and methods. 75

Results. 78

Discussion. 81

Acknowledgments. 85

Figures. 86

References 95

 

Chapter 4: Conclusions and perspectives. 99

Foreword. 100

EZH2, a potential TNBC therapeutic target 101

STING agonism and IL-29 induction are promising immunotherapeutic routes for human LUAD.. 104

References 109

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Health Sciences, Oncology Biology, Molecular Health Sciences, Immunology
Keyword	Cancer Immunotherapy Breast cancer Chemotherapy Neutrophils
Committee Chair / Thesis Advisor	Chrystal Mary Paulos, Emory University Periasamy Selvaraj, Emory University Curtis Henry, University of Colorado Anschutz School of Medicine Wei Zhou, Emory University Rabindra Tirouvanziam, Emory University

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