Author ORCID Identifier

https://orcid.org/0000-0001-8475-4982

Date of Graduation

5-2019

Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Gábor Balázsi, Ph.D.

Committee Member

Nicholas Navin, Ph.D.

Committee Member

Han Liang, Ph.D.

Committee Member

William Mattox, Ph.D.

Committee Member

Eric Brouzes, Ph.D.

Abstract

Drug resistance and metastasis remain obstacles to effective cancer treatment. A major challenge contributing to this problem is cellular heterogeneity. Even in the same environment, cells with identical genomes can display cell-to-cell differences in gene expression, also known as gene expression noise. Gene expression noise can vary in magnitude in a population or in fluctuation time scales, which is influenced by gene regulatory networks.

Currently, it is unclear how gene expression noise from gene regulatory networks contributes to drug survival outcomes in mammalian cells. An isogenic cell line with a noise-modulating genetic system tuned to the same mean is required. Additionally, how modulating endogenous mean gene expression and noise in living cells influences pro-survival metastatic state transitions remains unanswered.

To address these knowledge gaps, I implemented an exogenous synthetic biology approach to control noise for the drug resistance gene PuroR in drug survival while complementing with endogenous expression measurements of the pro-metastatic gene BACH1 as a correlate for metastatic survival. For exogenous control, I developed synthetic gene circuits in Chinese Hamster Ovary (CHO) cells based on positive and negative feedback that tune noise for PuroR at identical mean expression. At a decoupled noise point, isogenic cells were treated with various Puromycin concentrations. Evolution experiments revealed that noise hurts drug resistance during low drug dosage while facilitating resistance at a high Puromycin concentration. Drug adaptation for the low-noise gene circuit relied on intra-circuit mutations while the high-noise circuit did not and became re-sensitized to drug after removing circuit induction.

To implement the endogenous approach, I tagged the endogenous BACH1 gene with the mCherry fluorescent protein in six HEK293 clones. Molecular perturbations such as serum starvation and long-term hemin treatment altered mean fluorescence in at least one clone. Additionally, monitoring migration after cell wounding revealed increased non-uniform fluorescence at the wound edge. The increased mean fluorescence for the potentially bistable HEK293 clone 2C10 during hemin treatment may reflect altered BACH1 state transitions. Overall, noise enhanced the probability of cells to reach an expression level that confers survival during drug treatment while hemin perturbations may induce a pro-survival metastatic transition via BACH1 expression.

Keywords

Mammalian synthetic gene circuits, Positive and negative feedback, Gene expression noise, Drug resistance, Experimental evolution, CRISPR-Cas9-mediated gene knock-in, Endogenous expression of pro-metastatic BACH1 gene

Available for download on Monday, November 04, 2019

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