Student Theses and Dissertations

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Tavazoie Laboratory

Abstract

Metastatic disease is the major cause of death in all solid tumor cancers. Current therapeutic strategies fail to target metastasis as the genes and mechanisms that regulate this process remain poorly understood. Metastatic colonization is the final step of the metastatic cascade whereby cancer cells form a tumor at a distant site. This final step is the culmination of clonal evolution of cancer populations that results in a highly aggressive population with enhanced metastatic capacity and often presents clinically as numerous inoperable tumor nodules that lead to mortality. Characterization of the mechanisms that govern metastatic colonization at cellular and molecular levels is necessary for the prevention and treatment of metastatic disease in patients. The first half of this thesis presents work towards understanding mechanisms that mediate colorectal cancer colonization of the liver in order to guide novel therapeutic strategies. An in vivo large-scale RNAinterference screen was performed to identify genes required for liver colonization. Liver and red blood cell pyruvate kinase (PKLR) was identified as a driver of liver metastasis in experimental models. In patients, PKLR was found to be expressed at higher levels in liver metastases relative to primary colorectal cancer tumors and also overexpressed in the primary tumors of patients with metastatic disease. PKLR was found to promote cell survival in the tumor core and enhance survival during conditions of concurrent high cell density and low oxygen availability. Molecular studies revealed that PKL negatively regulates pyruvate kinase M2 (PKM2) enzymatic activity. By inhibiting cellular pyruvate kinase activity, PKLR allows for the diversion of metabolites towards glutathione generation—allowing for the maintenance of glutathione levels. Adequate glutathione levels appears critical for metastatic colonization as GCLC, the catalytic subunit of glutamatecysteine ligase and the rate-limiting enzyme for glutathione synthesis, was found to be similarly required for effective metastasis, associated in its expression with human liver metastatic progression, and could be therapeutically targeted to reduce metastatic colonization. These findings highlight the impact of metabolic regulation on cancer cell adaptation within the metastatic niche. The robust effects on liver metastatic colonization observed upon modulating this metabolic pathway suggest clinical potential for therapeutic targeting of PKLR or cellular glutathione synthesis in colorectal cancer. The second half of this thesis presents work towards an understanding of diversity generation in clonal populations as it benefits cancer evolution and metastatic colonization. Clonal human breast cancer subpopulations were isolated to allow for the identification of subpopulations that exhibit population-level phenotypic diversity. These high variability clonal subpopulations were found to be more proficient at metastatic colonization—consistent with a positive role for diversification capacity in cancer progression. Through single-cell RNA-sequencing, cell-to-cell transcript expression variability was identified as a defining feature of these subpopulations, extending to protein-level variability. Furthermore, spliceosomal machinery was identified as a gene set with high expression variability, suggesting a means by which variation could be transmitted to a global level. Engineered variable expression of the spliceosomal gene SNRNP40 promoted metastatic fitness, and this metastatic capacity was attributable to cells with low SNRNP40 expression. Clinically, low SNRNP40 expression is associated with metastatic relapse. These findings reveal that transcriptomic variability generation may serve as a mechanism by which cancer subpopulations achieve diversification of gene expression states, which allows for enhanced fitness under changing environmental pressures encountered during metastatic progression.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Available for download on Sunday, May 20, 2018

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