Regulation of Epithelial-Mesenchymal Transition via Matrix Stiffness and Histone Methyltransferases

Restricted (Penn State Only)
- Author:
- Sankhe, Chinmay
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 03, 2023
- Committee Members:
- Andrew Zydney, Major Field Member
Esther Gomez, Chair & Dissertation Advisor
Amir Sheikhi, Major Field Member
Sergei Grigoryev, Outside Unit & Field Member
Robert Rioux, Professor in Charge/Director of Graduate Studies - Keywords:
- Hyaluronic acid
atomic force microscopy
cytoskeleton
integrin-linked kinase
apoptosis
hydrogels
Hyaluronic Acid
Atomic Force Microscopy
Cytoskeleton
Apoptosis
Hydrogels
epigenetics
H3K9Me2
chromatin
Hippo Signaling
nuclear localization
biosensor - Abstract:
- Epithelial-Mesenchymal Transition (EMT) is a biological process that imparts dynamic regulatory changes in cellular behaviors, initiating conversion to a mesenchymal phenotype with cytoskeletal remodeling and enhanced motility. EMT is important during organ development and wound healing, but dysregulated EMT programs contribute to pathological contexts including fibrosis and cancer. Increased extracellular matrix stiffness is a hallmark of fibrosis and cancer, and previous studies have shown that the stiffness of the extracellular matrix modulates EMT-associated gene expression changes. Histone modifications, which are implicated in regulation of EMT, can impact chromatin organization thereby modulating transcription and repression of different genes. In particular, methylation at amino acid lysine (K) at the 9th position on histone H3 promotes repression of the epithelial marker E-cadherin in cancer cells. Methyltransferase enzymes such as G9a and SUV39H1 catalyze H3K9 methylation. While several reports have indicated abnormal expression of G9a and SUV39H1 in various types of cancers, there is a limited knowledge available on how matrix stiffness regulates methyltransferases in the context of the EMT process. We find that matrix stiffness and TGFβ1 signaling regulates H3K9 methylation markers, H3K9Me2 and H3K9Me3, along with regulating the levels of G9a, suggesting that matrix stiffness and TGFβ1 signaling can regulate chromatin dynamics. In addition, we find that the activity of G9a and SUV39H1 is essential for TGFβ1-induced EMT as inhibition of these methyltransferases results in attenuation of EMT-associated changes in response to stiffness. We identify the LATS-YAP signaling cascade as a possible mechanism by which G9a mediates TGFβ1-induced EMT in response to matrix stiffness. Furthermore, we find that inhibition of cell contractility can attenuate levels of G9a along with regulating H3K9 methylation with increasing matrix stiffness. Cumulatively, these results provide insights into molecular targets in the form of epigenetic signaling that can potentially be explored in the context of cancer and fibrosis. Cancer cells are thought to undergo mesenchymal-epithelial transition (MET) to form secondary tumors; however, there is a lack of in vitro platforms to study MET in cancer cells. Here, we focused on fabrication and characterization of a hyaluronic acid-based hydrogel platform with dynamic and tunable stiffnesses mimicking that of normal and tumorigenic mammary tissue to study the MET and EMT response in breast cancer cells. We find that breast cancer cells undergo dynamic changes in morphological properties with a decrease in cell spreading and elongation as the stiffness of the hydrogel gradually lowers with time. In addition, breast cancer cells transition towards an epithelial-like phenotype with dynamic softening of the matrix. Furthermore, we find that softening of the matrix also leads to a gradual decrease in integrin linked kinase (ILK) protein expression. These findings suggest that targeting the modulus of the tumor microenvironment may be a useful therapeutic approach for cancer. Overall, this dissertation elucidates mechanistically how extracellular matrix stiffness regulates the EMT process, in particular through regulation of methyltransferases and cell contractility. The findings suggest signaling molecules and pathways that can potentially be targeted for blocking EMT-associated functions in cancer and fibrosis.