DOI

10.17077/etd.ua71cjl0

Document Type

Dissertation

Date of Degree

Summer 2017

Access Restrictions

Access restricted until 08/31/2019

Degree Name

PhD (Doctor of Philosophy)

Degree In

Free Radical and Radiation Biology

First Advisor

Goswami, Prabhat C.

First Committee Member

Spitz, Douglas R.

Second Committee Member

Buettner, Garry R.

Third Committee Member

Domann, Frederick E.

Fourth Committee Member

Abel, E Dale

Abstract

Replicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan is defined as the duration during which quiescent normal cells retain their capacity to re-enter the proliferative cycle. This study investigates whether changes in metabolism occur during aging of quiescent normal human fibroblasts (NHFs) and the mechanisms that regulate these changes. Bioenergetics measurements were performed in quiescent NHFs from younger (newborn, 3-d, 5-m, and 1-y) and older (58-y, 61-y, 63-y, 68-y, and 70-y) donors as well as NHFs from the same individual at different ages (29-y, 36-y, and 46-y). Results show significant changes in cellular metabolism during aging of quiescent NHFs: old NHFs exhibit significant decreases in glycolytic flux and lactate levels, and increases in oxygen consumption rate (OCR) and ATP levels compared to young NHFs. Results from Seahorse Mito Stress Test show that old NHFs with a lower Bioenergetic Health Index (BHI) are more prone to oxidative stress compared to young NHFs with a higher BHI. The increase in OCR in old NHFs is associated with a shift in mitochondrial dynamics more towards fusion. Genetic knock-down of mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in old NHFs decreased OCR and shifted metabolism more towards glycolysis. Downregulation of MFN1 and OPA1 also suppressed the radiation-induced increase in doubling time of NHFs. These results suggest that a metabolic shift from glycolysis in young to mitochondrial respiration in old NHFs occurs during the chronological lifespan, and MFN1 and OPA1 regulate this process.

Age-associated metabolic reprogramming can also impact the age-related disease progression such as cancer. Recent evidence suggests a significant role of fibroblasts in pancreatic ductal adenocarcinoma (PDAC) stromal cellularity, metabolism, and therapy response. Considering PDAC being an age-related disease and a dismal 5-y survival of less than 9%, this study investigates whether stromal aging regulates PDAC progression. Results show that NHFs from older healthy individuals stimulate proliferation of PDAC cells compared to younger NHFs. Results from an in vivo study show that rate of tumor growth in xenografts of PDAC cells cultured with the old NHFs is significantly increased compared to the co-cultures of the young NHFs. In addition, decreased survival was also observed in mice carrying xenograft of co-culture of PDAC and the old NHFs compared to the young NHFs. Results from quantitative RT-PCR assays show that arachidonic acid lipoxygenase (ALOX12) expression decreased in PDAC, but increased in stromal fibroblasts in an age-dependent manner. Molecular inhibition of ALOX12 in the old NHFs suppressed PDAC proliferation in co-culture. These results show that aging of stromal fibroblasts aging promotes progression of PDAC, and identified ALOX12 and its metabolite 12-hydroxyeicosatetraenoic acid (12(S)-HETE) as critical regulators of PDAC proliferation.

Taken together, findings from this project demonstrate that age-associated metabolic reprogramming of NHFs from glycolysis in young to mitochondrial respiration in old regulates fibroblasts-induced stimulation of proliferation of human PDAC. Importantly, results from this study are anticipated to contribute to the development of novel approaches targeting stromal aging for cancer prevention and therapy response.

Keywords

aging, metabolism, MFN1, mitochondria, OPA1, respiration

Pages

xx, 139 pages

Bibliography

Includes bibliographical references (pages 113-139).

Copyright

Copyright © 2017 Jyung Mean Son

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