Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

Summer 9-1-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

David Piwnica-Worms

Abstract

Over the past decade several lines of research have indicated that DAPK3, a member of the serine/threonine death associated protein kinase (DAPK) family, plays an important role in various signaling pathways important to tissue homeostasis and mammalian biology. Considered to be a putative tumor suppressor, the molecular mechanisms by which DAPK3 exerts its tumor suppressor functions are not fully understood. Furthermore, unlike other DAPK family members, DAPK3 has received little attention regarding its physiological roles in vivo due to the lack of knockout animals. To address these gaps in our fundamental understanding of DAPK3 we

utilized the MCF10A 3D tumorigenesis model in vitro and generated a constitutive DAPK3 knockout mouse. Using the MCF10A 3D morphogenesis model we identified that loss of DAPK3 through lenti-viral mediated knockdown accelerates MCF10A acini proliferation and apoptosis while maintaining acini polarity relative to negative control. Furthermore, depletion of DAPK3 leads to enhanced growth factor-dependent mTOR activation and enlarged DAPK3 structures are uniquely sensitive to low doses of rapamycin treatment compared to negative control. Simultaneous knockdown of RAPTOR (a key mTORC1 component) rescues the augmented acinar size in DAPK3 depleted structures indicating an epistatic interaction. To identify the overall physiological contribution of DAPK3 we generated a constitutive DAPK3 knockout mouse using a gene trap embryonic stem cell line from the International Gene Trap

Consortium. Described herein we have identified that DAPK3 is vital for early mouse development and that the Dapk3 promoter exhibits spatio-temporal activity in the developing heart and nervous system in addition to the gastrointestinal myenteric plexus of adult mice.

Importantly, our data suggests that DAPK3 is expressed in the breast epithelia of adult mice and that potential ablation of DAPK3 expression can facilitate the development of breast cancer as

observed in primary patient biopsies. Our studies shed light on the growth inhibitory mechanisms of DAPK3 and provide direct evidence that DAPK3 plays an under-appreciated role in mouse development, warranting further studies.

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html

Permanent URL: http://dx.doi.org/10.7936/K70P0X26

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