SUMMARY Intense noise exposure causes hearing loss by inducing degeneration of spiral ganglia neurites that innervate cochlear hair cells. Nicotinamide adenine dinucleotide (NAD+) exhibits axon-protective effects in cultured neurons, however, its ability to block degeneration in vivo has been difficult to establish due to its poor cell permeability and serum instability. Here, we describe a strategy to increase cochlear NAD+ levels in mice by administering nicotinamide riboside (NR), a recently described NAD+ precursor. We find that administration of NR, even after noise exposure, prevents noise-induced hearing loss (NIHL) and spiral ganglia neurite degeneration. These effects are mediated by the NAD+-dependent mitochondrial sirtuin, SIRT3, since SIRT3-overexpressing mice are resistant to NIHL and SIRT3 deletion abrogates the protective effects of NR and expression of NAD+ biosynthetic enzymes. These findings reveal that administration of NR activates a NAD+-SIRT3 pathway that reduces neurite degeneration caused by noise exposure.

BACKGROUND Several sirtuin family members (SIRT1-7), evolutionarily conserved NAD-dependant deacetylases, play an important role in carcinogenesis. However, their role in oral cancer has not yet been investigated. Therefore, the aim of this study was to investigate whether sirtuins play a role in oral cancer carcinogenesis. METHODS We examined the expression levels of all sirtuins in several oral squamous cell carcinoma (OSCC) cell lines compared to normal human oral keratinocytes, and found SIRT3 was highly expressed. Therefore, tissue microarrays were used to evaluate the clinical relevance of this overexpression. SIRT3 downregulation in OSCC cell proliferation and survival was then investigated and analyzed by cell proliferation and cell viability assays. Ionizing radiation and cisplatin were used to investigate whether SIRT3 downregulation can increase the sensitivity of OSCC to both treatments. To further assess the in vivo role of SIRT3 in OSCC carcinogenesis, we used a floor-of-mouth oral cancer murine model to study the effect of SIRT3 downregulation on OSCC tumor growth in immunodeficient mice. RESULTS We show for the first time that SIRT3 is overexpressed in OSCC in vitro and in vivo, compared to other sirtuins. Downregulation of SIRT3 inhibited OSCC cell growth and proliferation, and increased its sensitivity to radiation and cisplatin treatments in vitro. SIRT3 downregulation reduced tumor burden in vivo. CONCLUSIONS Our findings reveal a novel role for SIRT3 in oral cancer carcinogenesis as a promoter of cell proliferation and survival, thus implicating SIRT3 as a new potential therapeutic target to treat oral cancer.

The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.cancer signaling | single molecule imaging | superresolution optical microscopy T he RAF serine/threonine protein kinase is a component of the three-tiered MAPK signaling pathway that regulates cell growth and many other essential biological processes (1, 2). In normal cells, extracellular mitogenic stimuli are transmitted to the nucleus via the receptor-RAS-RAF-MAPK cascade (2). Abnormal activation of this pathway is a central event in many human cancers and results from activating mutations in BRAF itself or in upstream factors (such as the RAS genes) (3, 4). As the RAS proteins so far are intractable pharmacologic targets (5), attention has shifted to development of small-molecule RAF inhibitors (RAFis) as antitumor therapeutic agents (6). To date, RAFi clinical efficacy has been demonstrated only for BRAF V600E melanoma (6-8). In tumors with WT BRAF or mutant RAS, most RAFis paradoxically promote growth, at times malignant in nature (6). Moreover, BRAF mutant melanomas that are initially sensitive to RAFi rapidly become resistant by using a variety of compensatory mechanisms including RAF isoform switching and activation of other pathways including RTKs, RAS, or PI3K (9). In addition, some RAFis (e.g., vemurafenib) accelerate the occurrence of secondary squamous cell carcinomas (10).Several lines of investigation suggest that multimer formation plays an important role in RAF activation and tumor responses to RAFi (11-16). RAF dimerization-mediated signaling was first suggested by the observation that ar...