Date of Award

5-1-2017

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Rao, Krishna

Abstract

The RAB guanosine triphosphates (RAS-related in brain) belong to the Ras superfamily of GTPases, and loss of RAB 25 expression has been reported in a number of breast cancer cases containing H-Ras point mutations, particularly triple negative breast cancers (TNBC), one of the most aggressive subtypes of breast cancer and associated with a poor prognosis. The mechanism involved in the progression of these tumors is poorly understood. In this study, we are trying to understand if loss of RAB25 expression in Human Mammary Epithelial Cell (HMEC) lines co-operates with H-Ras mutations and contributes to tumorigenesis. HMEC were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, catalytic subunit of the telomerase enzyme that permits the cells to exceed the Hayflick Limit and become immortal. We have found that with loss of RAB25 and over expression of mutant H-Ras61L, immortal HMEC undergo transformation. We have looked into the co-operativity between loss of Rab25 and H-Ras61L mutant by in-vitro studies to show their anchorage independent growth and increased ability to migrate. Furthermore, cells express low CD24, high CD44, and very low levels of Claudin indicating that cells acquire stem-like properties upon transformation. Loss of RAB25 and over-expression of H-ras61L resulted in increased expression of transcription markers Snail and Slug that drive these cells to lose E-cadherin and undergo Epithelial Mesenchymal Transition (EMT). This study shows that loss of RAB25 and over-expression of mutant H-Ras can transform HMEC and give rise to mesenchymal stem-like tumors. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker in the prognosis of Claudin-low type of TNBC.

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