Breast cancer metastases cause significant patient mortality. During metastases, cancer cells use autophagy, a catabolic process to recycle nutrients via lysosomal degradation, to overcome nutritional stress for their survival. The Runt-related transcription factor, Runx2, promotes cell survival under metabolic stress, and regulates breast cancer progression and bone metastases.Here, we identify that Runx2 enhances autophagy in metastatic breast cancer cells. We defined Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide molecular insights into the role of autophagy in metastatic cancer cells. K E Y W O R D Sα-tubulin, autophagy, breast cancer, LC3B, metastasis, Runx2

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.

Primary, or familial, hemophagocytic lymphohistiocytosis (P-HLH) is a rare inherited autosomal-recessive immune deficiency which generally manifests during infancy or early childhood. Recent literature suggests an increased number of reports of late-onset P-HLH, especially in association with infection and underlying malignancy. The authors describe a case of subcutaneous T-cell lymphoma in a 8-year-old child that was complicated by primary, perforin-deficient HLH. In contrast, we examined retrospective data for 19 cases of late-onset P-HLH with available treatment data and compared the results of conservative medical therapy with hematopoietic stem cell transplant (HSCT) postremission therapy. Our patient displayed compound heterozygous mutations in PRF1 that have not been described in the literature previously: allele 1 [c.786_801del(p.Gln263fs)] and allele 2 [c.886T>C(p.Tyr296His)]. Of the 19 cases analyzed, 14 achieved remission. Postremission, 7 of 14 (50%) received HSCT and were reported alive at a median time of 24 months, 5 of 14 (36%) received medical therapy and were reported alive at a median time of 24 months, and 2 of 14 (14%) received medical therapy and died at a median of 73 months postremission. Our retrospective literature review suggests that some patients can survive late-onset, perforin-deficient, P-HLH without the potential lifelong risks of HSCT when in the first remission.