Role of G1p3-induced Tubulin Acetylation Confers Metastatic Potential in Breast Cancer Cells

Swati Balluri

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Date of Award

Summer 2016

Abstract

The overall objective of this study is to elucidate the role of G1P3 in breast cancer metastasis. Metastatic breast cancer results in approximately 90% of the breast cancer related deaths. Therefore, delineating mechanisms of metastasis may help to prevent and reverse the metastasis. Although G1P3 is an antiapoptotic protein in mitochondria, cytoskeletal elements are remodeled in G1P3 expressing cells (MCF-7G1P3). Additionally, relative to control (MCF-7vector) cells, MCF-7G1P3 cells have significantly higher levels of mitochondrial reactive oxygen species (mtROS). Since high levels of mtROS are suggested to promote microtubule acetylation, we hypothesized that GlP3-induced mtROS would increase tubulin acetylation to confer breast cancer metastatic potential. This hypothesis was evaluated by immunoblot, which showed a marked increase in tubulin levels in MCF-7G1P3 cells relative to MCF-7vector cells. This was confirmed by immunostaining, which also identified altered microtubule organization in MCF-7G1P3 cells relative to MCF-7vector. Moreover, the acetylation of tubulin at lysine 40 was significantly higher in MCF-7G1P3 compared to MCF-7vector cells (p = 0.0223). To determine what role mtROS induced by G1P3 played on tubulin acetylation, N-acetyl cysteine (NAC, a ROS scavenger) was utilized to reduce ROS levels and to study its impact on tubulin acetylation. With NAC treatment, western blot analysis showed that acetylation of tubulin markedly decreased in MCF-7G1P3 cells compared to vector control. Also, immunostaining confirmed that NAC treatment significantly decreased acetylation in MCF-7G1P3 treated cells (p = 0.0213), further demonstrating the role of G1P3-induced mtROS mediation of tubulin-acetylation. As mtROS levels are high in MCF-7G1P3 cells, NAD (a byproduct of ETC) was expected to be high. As NAD acts as a cofactor for class III Histone deacetylases – sirtuins, the levels of NAD/NADH were evaluated to understand the mechanism of G1P3-induced tubulin acetylation. Relative to MCF-7vector, MCF-7G1P3 cells have significant increase in NAD/NADH levels (p = 0.0128). To study the role of G1P3-induced tubulin acetylation in promoting metastasis, wound healing assay was performed, which identified that MCF-7G1P3 cells migrated significantly faster than MCF-7vector cells (1.45 fold, p = 0.0441). Additionally, to further confirm the role of G1P3-induced tubulin acetylation to confer metastatic potential in breast cancer cells, BML278 (a SIRT activator) was used. With BML278 treatment, the migration in MCF-7G1P3 cells significantly decreased (p = 0.0152) further suggesting the role for G1P3-induced tubulin acetylation in metastasis. Furthermore, HDAC assay was performed to determine the deacetylation activity in MCF-7vector and MCF-7G1P3 cells, which identified that G1P3 expressing cells have marked decrease in deacetylase activity, relative to control. Based on these results, we suggest that G1P3 promotes metastasis by inducing tubulin acetylation.

Advisor

Venu Cheriyath

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Biology | Life Sciences

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