Evidence for a pro-oncogenic role of Sirtuin 6 in breast tumorigenesis

Background: Sirtuin 6 (SIRT6) is a member of the sirtuin family, NAD+-dependent deacetylases with key roles in cell metabolism, DNA repair and inflammation. High SIRT6 levels in breast tumors confer an adverse prognosis. However, the underlying mechanism for such observations have remained unclear in so far. Here I sought to define the effect of a heterozygous Sirt6 deletion on polyoma middle T antigen-induced mouse mammary tumorigenesis and to establish the biochemical and molecular effects of overexpressing vs. reducing SIRT6 in different breast cancer (BC) models. Methods: SIRT6 was overexpressed in either wild type or catalytically inactive (H133Y) form, or silenced in BC cell lines (MDA-MB-231 and MCF7), and we monitored oxygen consumption rate, mitochondrial complex I, III, IV, and ATP synthase activity, cell migration and invasion in Matrigel and in transwell assays, matrix metalloproteinase 9 (MMP9) expression and intracellular Ca2+ concentration ([Ca2+]i). In vivo, we monitored the growth of MDA-MB-231 xenografts in which SIRT6 was silenced vs. control tumors. In addition, we crossed Sirt6+/- mice with MMTV-PyMT+/- mice and comparatively monitored tumor latency and overall survival in MMTV-PyMT+/-;Sirt6+/- vs. MMTV-PyMT+/-;Sirt6+/+ mice. Results: In cultured BC cell lines, overexpression of a catalytically active SIRT6 (but not of the catalytically inactive isoform) boosted OXPHOS and the ATP/AMP ratio. Opposite effects were obtained by SIRT6 silencing. Modulating SIRT6 profoundly affected MMP9 expression and [Ca2+]i. Namely, in MDA-MB-231, SIRT6 overexpression increased, while SIRT6 silencing reduced MMP9 production. [Ca2+]i was increased in WT-SIRT6 overexpressing MDA-MB-231 and such an effect appeared to reflect Ca2+ freeing from its thapsigargin-sensitive stores. Consistent with these data, SIRT6-overexpressing MDA-MB-231 were more invasive than their control cells in vitro assays. In vivo, subcutaneous xenografts of SIRT6-silenced MDA-MB-231 cells were found to grow more slowly than the control tumors. MMTV-PyMT+/-;Sirt6+/- mice exhibited a markedly increased tumor latency and an increased overall survival as compared to the control MMTV-PyMT+/-;Sirt6+/+ animals. The metabolic features of the tumor masses isolated from MMTV-PyMT+/-;Sirt6+/- mice resembled those observed in BC cell lines with silenced SIRT6, showing decrease mitochondrial complexes activity and impaired energy status. The anticancer effects of Sirt6 heterozygous deletion did not reflect reduced glucose levels in Sirt6+/- mice, as the latters had normal blood glucose concentrations. Conclusions: Our data show that reducing Sirt6 levels has significant antitumor activity in in vivo BC models. SIRT6 enhances OXPHOS and energy status in BC cells. In addition, by virtue of its ability to enhance MMP9 expression and [Ca2+]i, SIRT6 could be a potential target for countering invasion and metastasis. Future studies should assess which molecular features predict the potential benefit of SIRT6 inhibition in BC and test the anticancer activity of SIRT6 inhibitors in BC models.