Identifying and functionally characterizing transcriptional drivers of esophageal cancer progression for nucleic acid-based therapeutic development

Esophageal cancer, a cancer of poor prognosis, arises as either squamous or adenomatous forms, bearing many hallmarks of their tissue of origin. Esophageal squamous cell carcinoma (ESCC) originates from squamous epithelial cells, whereas esophageal adenocarcinoma (EAC) arises from the columnar cells of a precancerous lesion known as Barrett’s Esophagus (BE) following the development of low grade dysplasia (LGD) and subsequent high grade dysplasia (HGD). Furthering our understanding of the biological processes that drive the emergence of these distinct variants will be critical to develop novel targeted therapies and improve outcomes for esophageal cancer patients.

One important biological aspect of cancer progression is the role of nuclear proteins known as transcription factors (TFs). TFs act as master regulators of cellular function and communication by determining which genes a given cell should utilize, activate, or deactivate. In ESCC and EAC, squamous cell (in ESCC) or columnar cell (in EAC) -associated TFs often become aberrantly activated, indicating that divergent programs of gene activation occur in these cancer variants. Furthermore, the overarching cancer-promoting programs engaged by these TFs and their associates, as well as their mechanisms of cancer facilitation through downstream target activation, remain to be full elucidated. Identifying the divergent TF-driven programs activated in these cancers and their consequential impacts on oncogenesis and metastasis could lead to the development of new therapeutic interventions for esophageal cancer patients.

In this project, we aim to identify and characterize the functional impact of the transcriptional programs that drive esophageal cancer progression, with the goal of improving our understanding of the disease’s biology and providing critical insights that could guide innovative therapeutic targeting of oncoproteins.

The proposed project has two primary aims:

Aim 1: Identify functionally relevant TFs driving progression of esophageal cancer variants
To identify functionally relevant TFs that drive ESCC and EAC progression, we will develop and employ a functional DNA barcode-based high-throughput reporter assay to identify TFs that are differentially activated across various esophageal cancer cells and conditions. We will complement this with comparative bioinformatics analysis of ESCC and EAC genomic datasets to identify TFs with differential DNA-binding and regulatory activity between the two cancer types. This integrative approach will enable us to pinpoint key transcriptional regulators specific to ESCC and EAC and shed light on their distinct molecular drivers.

Aim 2: Investigate the downstream targetable gene expression programs regulated by candidate TFs identified in aim 1
To understand how the transcription factors identified in Aim 1 contribute to either ESCC or EAC progression including through the stages of LGD and HGD, it is crucial to elucidate the downstream gene expression programs that they regulate. This aim will focus on mapping the transcriptional and genomic targets of these TFs to determine their functional relevance to esophageal cancer. To do this, we will perform CRISPR gene knockouts of candidate TFs and perform subsequent RNA-sequencing on the wildtype cells compared to knockout cells, to examine genes differentially expressed. By characterizing and contrasting these downstream pathways between esophageal cancer variants, we can identify new molecular mechanisms that drive EAC or ESCC and discover additional therapeutic targets.

During the internship, the MITACS fellow will develop a diverse set of skills and competencies that will support them as a researcher and enhance their professional development. The fellow will further develop their technical proficiencies in advanced molecular and cellular biology techniques, such as RNA sequencing, high-throughput screening methods such as massively parallel reporter assays, and ultimately CRISPR gene editing. Through this internship, the fellow will also advance their project management skills, as they learn to coordinate their research activities between academic and industry settings, set timelines, and manage deliverables. They will also enhance their critical thinking and problem-solving abilities through troubleshooting experiments and interpreting complex data sets. Furthermore, they will have the opportunity to build their professional network within both the academic and industry circles, as well as improve their communication skills through presentations and collaborative discussions with diverse groups. Overall, this opportunity will enable the fellow to develop a comprehensive skill set that integrates academic research with real-world applications.

This project will support Promirin Therapeutics Ltd’s mission of developing targeted cancer therapeutics. By identifying TFs that drive the progression of esophageal cancer, as well as their downstream targets, the research will lead to a better understanding of the disease at a molecular level. This project will help establish the knowledge based required for Promirin to apply their targeted nucleic-acid based inhibitory technology in the context of this cancer type. Lastly, the project will support the Promirin’s broader goals of promoting innovative research and improving patient outcomes.