Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Robert J. Gillies, Ph.D.

Co-Major Professor

Shari Pilon-Thomas, Ph.D.

Committee Member

Gina DeNicola, Ph.D.

Committee Member

Andriy Marusyk, Ph.D.

Committee Member

Patricia McDonald, Ph.D.

Committee Member

Paul Stewart, Ph.D.

Committee Member

Pawel Swietach, Ph.D.

Keywords

Aerobic fermentation, Low pH, Warburg phenotype, Tumor evolution

Abstract

In the early 20th century, Nobel laureate Otto Warburg made the observation that cells of a carcinoma had considerably higher glycolytic metabolism and considerably lower oxidative metabolism compared to cells of a normal tissue. He postulated that within this observation was the key to deciphering the differences between malignant and normal tissue. It is now well established that tumors of the breast are unequivocally acidic, caused by an abnormal amount of aerobic glycolysis, colloquially known as the Warburg effect. Over the last decades, our group, led by Dr. Robert J. Gillies, has set out to characterize the causes and consequences of this acidity in cancer. Major strides have been made in understanding how the acidic environment is developed, and the effects it has on the progression of the tumor. Important questions still exist in understanding how this acidity affects the phenotype of cellular populations within the tumor, how we can effectively target this acidic phenotype in the treatment of cancer, and how the glycolytic phenotype of cancer cells is regulated by acidity. The aim of this work was to contribute to the understanding of the two questions posited in the preceding text.

Chapter 3: Early ducts of breast tumors are unequivocally acidic. High rates of glycolysis combined with poor perfusion lead to congestion of acidic metabolites in the tumor microenvironment, and pre-malignant cells must adapt to this acidosis to thrive. Adaptation to acidosis selects cancer cells that can thrive in harsh conditions and are capable of outgrowing the normal or non-adapted neighbors. This selection is usually accompanied by phenotypic changes. Epithelial mesenchymal transition (EMT) is one of the most important switches correlated to malignant tumor cell phenotype and has been shown to be induced by tumor acidosis. New evidence shows that the EMT switch is not a binary system and occurs on a spectrum of transition states. During confirmation of the EMT phenotype, my results demonstrated a partial EMT phenotype in the acid-adapted cell population. Using RNA sequencing and network analysis we found 10 dysregulated network motifs in acid-adapted breast cancer cells playing a role in EMT. The further integrative analysis of RNA sequencing and SILAC proteomics resulted in recognition of S100B and S100A6 proteins at both the RNA and protein level. Higher expression of S100B and S100A6 was validated in vitro by Immunocytochemistry (IHC). I further validated our finding both in vitro and in patients' samples by IHC analysis of Tissue Microarray (TMA). Correlation analysis of S100A6 and LAMP2b as a marker of acidosis in each patient from Moffitt TMA approved the acid related role of S100A6 in breast cancer patients. Also, ductal carcinoma in situ (DCIS) patients with higher expression of S100A6 showed lower survival compared to lower expression. We propose essential roles of acid adaptation in cancer cells EMT process through S100 proteins such as S100A6 that can be used as therapeutic strategy targeting both acid-adapted and malignant phenotypes.

Chapter 4: Evolutionary dynamics can be used to control cancers when a cure is not clinically considered to be achievable. Understanding Darwinian intratumoral interactions of microenvironmental selection forces can be used to steer tumor progression towards a less invasive trajectory. Here, we approach intratumoral heterogeneity and evolution as a dynamic interaction among subpopulations through the application of small, but selective biological forces such as intracellular pH (pHi) and/or extracellular pH (pHe) vulnerabilities. Increased glycolysis is a prominent phenotype of cancer cells under hypoxia or normoxia (Warburg effect). Glycolysis leads to an important aspect of cancer metabolism: reduced pHe and higher pHi. We recently showed that decreasing pHi and targeting pHi sensitive enzymes can reverse the Warburg effect (WE) phenotype and inhibit tumor progression. Herein, I used diclofenac (DIC) repurposed to control MCT activity, and koningic acid (KA) that is a GAPDH partial inhibitor, and observed that one could control the subpopulation of cancer cells with WE phenotype within a tumor in favor of a less aggressive phenotype without a WE to control progression and metastasis. In 3D spheroid co-cultures, I showed that our strategy can control the growth of more aggressive MDA-MB-231 cells, while sparing the less aggressive MCF7 cells. In an animal model, we show that our approach can reduce tumor growth and metastasis. We thus propose that evolutionary dynamics can be used to control tumor cells' clonal or sub-clonal populations in favor of slower growth and less damage to patients. We propose that this can result in cancer control for tumors where cure is not an option.

In total, this work has provided knowledge to the field by better describing how cellular populations of breast cancer cells adapt to an acidic environment, and how fermentative glycolysis can be exploited to control breast cancer populations.

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