Date of Award

8-1-2020

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (x, 113 pages) : illustrations (some color)

Dissertation/Thesis Chair

JoEllen Welsh

Committee Members

Martin Tenniswood, Douglas Conklin, Valerie Bolivar, Ramune Reliene

Keywords

Breast Cancer, HAS2, Hyaluronic Acid, STAT3, TGFβ1, TNFAIP6, Hyaluronic acid, Transforming growth factors-beta, Breast, Transcription factors, Cellular signal transduction, Transforming Growth Factor beta, Transcription Factors, Signal Transduction

Subject Categories

Cell Biology | Molecular Biology

Abstract

The studies described here explored the role of Transforming Growth Factor Beta-1 (TGFβ1) and Signal Transducer and Activator of Transcription 3 (STAT3) as potential regulators of the Hyaluronic Acid (HA) synthesis and signaling pathway in human mammary cells. Our results support previous findings in which TGFβ1, a well characterized driver of the epithelial-mesenchymal transition (EMT) has been shown to regulate HA synthesis and signaling. Interrogation of The Cancer Genome Atlas (TCGA) indicated HAS2 expression positively correlated with TGFβ1 mRNA expression in breast cancer patients and in breast cancer cell lines. RT-qPCR experiments were used to measure the expression of the genes involved in hyaluronan synthesis (HAS1, HAS2, and HAS3) including the long non-coding RNA, HAS2-AS1, which has been shown to enhance HAS2 transcription. Results demonstrated a significant increase in the expression of both HAS2 and HAS2-AS1, in human mammary epithelial cells overexpressing TGFβ1 (HMLE-TGFβ1 cells). To examine whether TGFβ1 regulated the degradation of HA we measured the mRNA expression of three hyaluronidases, HYAL1, HYAL2 and HYAL3. Results indicated no significant differences in the expression of HYALs. HMLE-TGFβ1 cells expressed higher GFPT2 which encodes for the rate-limiting enzyme of the Hexosamine Biosynthetic Pathway (HBP) that generates hexoses required for HA synthesis. Similarly, HAS2 and TGFβ1 mRNA expression correlated strongly with the expression of GFPT2 in breast cancer patients and breast cancer cells. The increased expression of HAS2 correlated with increased HAS2 protein abundance and HA secretion by HMLE-TGFβ1 cells, however there was no evidence for up-regulation. Importantly, treatment of HMLE-TGFβ1 cells with the HA inhibitor 4-MU reduced migration rate and mammosphere size.

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