Abstract 43: Regulation of autophagy in bone metastatic breast cancer cells
Authors: Ahmad H. Othman, Manish Tandon, Vivek Ashok, Marcus Winogradzki, Gary Stein, Jitesh Pratap
Journal: Cancer Research (2018)

Abstract Bone metastasis of breast cancer is a significant cause of patient mortality. Recent studies suggest that metastatic cancer cells induce autophagy to survive metabolic stress. During autophagy, cytoplasmic components and damaged organelles are captured by autophagosomes followed by lysosomal fusion and degradation, releasing metabolites as energy sources to meet metabolic demands. Although the components of the autophagy pathway have been well characterized, the regulatory mechanisms of autophagy in metastatic cancer cells remain unknown. Previously, we have shown that Runt-related transcription factor-2 (Runx2) promotes cell survival, bone metastasis, and osteolysis. Using a bone metastatic isogenic variant of breast cancer MDA-MB-231, we examined levels of the autophagosome specific marker LC3B to define the regulation of autophagy during bone metastasis. Additionally, we examined whether Runx2 regulates autophagy for increased cell survival in the bone microenvironment. Microscopic and biochemical studies showed elevated levels of autophagic flux among bone derived cells relative to parental breast cancer cells. Interestingly, we also observed that Runx2 enhanced the turnover of autophagic vesicles while Runx2 silencing resulted in accumulation of vesicles due to reduced turnover. Interestingly, Runx2 knockdown or inhibition of autophagy in bone derived cells increased AMPK levels suggesting higher levels of cellular stress as a consequence of impaired autophagy. In addition to AMPK activity, MAP kinase mutations have been demonstrated to result in constitutive activation of the autophagy pathway. Treatment with the MEK inhibitor PD184161 resulted in accumulation of LC3B-II in control cells. Furthermore, Runx2 knockdown in bone derived cells display lower levels of ERK activity relative to controls. These results suggest that Runx2/ERK signaling is critical for autophagy in metastatic breast cancer cells. Our mechanistic studies revealed that Runx2 promotes autophagy by increasing acetylation of α-tubulin sub-units of microtubules and enhancing trafficking of autophagic vesicles. Introduction of a mutant α-tubulin construct incapable of being acetylated resulted in the accumulation of autophagic vesicles in control cells, similar to silencing of Runx2. Inhibition of autophagy resulted in decreased adhesion, migration, and survival of Runx2 knockdown cells. Furthermore, analysis of LC3B protein in clinical breast cancer specimens and tumor xenografts revealed significant association between high Runx2 and low LC3B protein levels. Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide insights into the role of autophagy in bone metastatic breast cancer cells. Citation Format: Ahmad H. Othman, Manish Tandon, Vivek Ashok, Marcus Winogradzki, Gary Stein, Jitesh Pratap. Regulation of autophagy in bone metastatic breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 43.