Author ORCID Identifier

0000-0002-2851-9272

Date of Graduation

5-2017

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Dr Anil K Sood

Committee Member

Dr Gary Gallick

Committee Member

Dr Anirban Maitra

Committee Member

Dr Robert Dantzer

Committee Member

Dr. Peiying Yang

Abstract

Objectives: Catecholamine-mediated effects driven by elevated adrenergic signaling are known to increase tumor growth and metastasis by direct effects on tumor cells. However, knowledge of effects of adrenergic signaling on other cells such as cancer-associated fibroblasts (CAFs) within the tumor microenvironment is limited. We hypothesize that adrenergic signaling and norepinephrine can accelerate the induction of CAF-phenotype to promote inflammation and metastasis.

Methods: Ingenuity Pathway Analysis and NetWalker were used to assess gene expression data from patients with known CESD (Center for Epidemiological Studies-Depression)-score alongside microdissected CAFs from primary ovarian cancer. Tumor samples from mice exposed to daily restraint-stress or non-stressed controls were assessed for alpha-smooth muscle actin (a-SMA) expression. Normal fibroblasts (NoF 151) were treated with conditioned media from non-treated and norepinephrine (NE) treated Skov3 and HeyA8 cells and analyzed for induction of CAF-phenotype by a-SMA expression. Gene and protein expression of cytokines and changes in migratory potential of fibroblasts were assessed.

Results: Among the significantly upregulated genes (fold-change >2) in patients with known CESD-score, 34 genes overlapped with microdissected CAF data. Among them are several networks involved in extracellular matrix (ECM) and inflammatory response. Restraint stress was associated with significantly increased levels of α -SMA, a marker for CAFs by both intensity and distribution in Skov3, HeyA8 and ID8 tumors. These increases in α-SMA were abrogated when mice were treated with broad beta-blocker propranolol during restraint stress in HeyA8 and Skov3 model. Further bioinformatics analysis of these datasets combined with gene array data from conditioned normal fibroblasts showed collagens are the most important biological downstream effectors for the adrenergic induced CAF phenotype. Consistent with the bioinformatics analyses, there was increased collagen (assessed by Trichrome stain) in the tumors from restraint-stress animals, and these were abrogated by propranolol treatment. Upstream bioinformatics analysis of CAF mediators showed cancer-cell derived INHBA (inhibin beta A, a member of the TGF-b family) can mediate the induction of CAF-phenotype upon adrenergic stimulation. Silencing INHBA in tumor cells decreased stress-induced tumor growth in Skov3 orthotropic model as well as decreasing levels of CAFs and collagen in tumors.

Conclusion: Sustained adrenergic stimulation results in significant increases in the CAF content in-vivo and accelerates conversion of normal fibroblasts to CAFs in vitro (fold change-2 fold, p


Keywords

Chronic Stress, catecholamines, cancer-associated fibroblasts, inhibin beta A, tumor stroma

Available for download on Wednesday, April 18, 2018

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