Nonmonotone invasion landscape by noise-aware control of metastasis activator levels.

A major pharmacological assumption is that lowering disease-promoting protein levels is generally beneficial. For example, inhibiting metastasis activator BACH1 is proposed to decrease cancer metastases. Testing such assumptions requires approaches to measure disease phenotypes while precisely adjusting disease-promoting protein levels. Here we developed a two-step strategy to integrate protein-level tuning, noise-aware synthetic gene circuits into a well-defined human genomic safe harbor locus. Unexpectedly, engineered MDA-MB-231 metastatic human breast cancer cells become more, then less and then more invasive as we tune BACH1 levels up, irrespective of the native BACH1. BACH1 expression shifts in invading cells, and expression of BACH1's transcriptional targets confirm BACH1's nonmonotone phenotypic and regulatory effects. Thus, chemical inhibition of BACH1 could have unwanted effects on invasion. Additionally, BACH1's expression variability aids invasion at high BACH1 expression. Overall, precisely engineered, noise-aware protein-level control is necessary and important to unravel disease effects of genes to improve clinical drug efficacy.

Does transcriptional heterogeneity facilitate the development of genetic drug resistance?

Non-genetic forms of antimicrobial (drug) resistance can result from cell-to-cell variability that is not encoded in the genetic material. Data from recent studies also suggest that non-genetic mechanisms can facilitate the development of genetic drug resistance. We speculate on how the interplay between non-genetic and genetic mechanisms may affect microbial adaptation and evolution during drug treatment. We argue that cellular heterogeneity arising from fluctuations in gene expression, epigenetic modifications, as well as genetic changes contribute to drug resistance at different timescales, and that the interplay between these mechanisms enhance pathogen resistance. Accordingly, developing a better understanding of the role of non-genetic mechanisms in drug resistance and how they interact with genetic mechanisms will enhance our ability to combat antimicrobial resistance. Also see the video abstract here: https://youtu.be/aefGpdh-bgU.

Network of mutually repressive metastasis regulators can promote cell heterogeneity and metastatic transitions.

The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistable-like behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.

Advancing Antimicrobial Resistance Research Through Quantitative Modeling and Synthetic Biology.

Antimicrobial resistance (AMR) is an emerging global health crisis that is undermining advances in modern medicine and, if unmitigated, threatens to kill 10 million people per year worldwide by 2050. Research over the last decade has demonstrated that the differences between genetically identical cells in the same environment can lead to drug resistance. Fluctuations in gene expression, modulated by gene regulatory networks, can lead to non-genetic heterogeneity that results in the fractional killing of microbial populations causing drug therapies to fail; this non-genetic drug resistance can enhance the probability of acquiring genetic drug resistance mutations. Mathematical models of gene networks can elucidate general principles underlying drug resistance, predict the evolution of resistance, and guide drug resistance experiments in the laboratory. Cells genetically engineered to carry synthetic gene networks regulating drug resistance genes allow for controlled, quantitative experiments on the role of non-genetic heterogeneity in the development of drug resistance. In this perspective article, we emphasize the contributions that mathematical, computational, and synthetic gene network models play in advancing our understanding of AMR to discover effective therapies against drug-resistant infections.

Role of network-mediated stochasticity in mammalian drug resistance.

A major challenge in biology is that genetically identical cells in the same environment can display gene expression stochasticity (noise), which contributes to bet-hedging, drug tolerance, and cell-fate switching. The magnitude and timescales of stochastic fluctuations can depend on the gene regulatory network. Currently, it is unclear how gene expression noise of specific networks impacts the evolution of drug resistance in mammalian cells. Answering this question requires adjusting network noise independently from mean expression. Here, we develop positive and negative feedback-based synthetic gene circuits to decouple noise from the mean for Puromycin resistance gene expression in Chinese Hamster Ovary cells. In low Puromycin concentrations, the high-noise, positive-feedback network delays long-term adaptation, whereas it facilitates adaptation under high Puromycin concentration. Accordingly, the low-noise, negative-feedback circuit can maintain resistance by acquiring mutations while the positive-feedback circuit remains mutation-free and regains drug sensitivity. These findings may have profound implications for chemotherapeutic inefficiency and cancer relapse.