Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. Histone chaperones are regulators of this process by preventing formation of non-nucleosomal histone–DNA complexes. Aprataxin and polynucleotide kinase like factor (APLF) is a non-homologous end-joining (NHEJ) DNA repair factor that possesses histone chaperone activity in its acidic domain (APLFAD). Here, we studied the molecular basis of this activity using biochemical and structural methods. We find that APLFAD is intrinsically disordered and binds histone complexes (H3-H4)2 and H2A-H2B specifically and with high affinity. APLFAD prevents unspecific complex formation between H2A-H2B and DNA in a chaperone assay, establishing for the first time its specific histone chaperone function for H2A-H2B. On the basis of a series of nuclear magnetic resonance studies, supported by mutational analysis, we show that the APLFAD histone binding domain uses two aromatic side chains to anchor to the α1–α2 patches on both H2A and H2B, thereby covering most of their DNA-interaction surface. An additional binding site on both APLFAD and H2A-H2B may be involved in the handoff between APLF and DNA or other chaperones. Together, our data support the view that APLF provides not only a scaffold but also generic histone chaperone activity for the NHEJ-complex.

Triple negative breast cancer, characterised by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER-2), has a poor prognosis mostly due to increased rate of distant metastases 1,2 . During the process of metastasation, cancer cells in primary tumour locally invade the tumour-associated stroma, detach from the invasion front of the tumour, and enter the lymphatic and/or blood vessels. Circulating cancer cells ultimately migrate through the capillary wall in distant tissues, re-attach to the extracellular matrix, and proliferate in a new microenvironment 3 . Once cancer cells detach from the main tumour mass, they must resist anoikis, a programmed cell death induced by extracellular matrix detachment 4 . MDA-MB-231 cells, the most commonly used in vitro model of triple negative breast cancer 5 , are highly metastatic and tumorigenic 5 . They form colonies in an anchorage-independent condition 6 , and are resistant to anoikis 7 . Albeit breast cancer cells must detach from extracellular matrix in order to metastasise in vivo [8][9][10][11] , floating MDA-MB-231 cells in vitro are commonly thought to be dead. Only a few studies investigated the viability of floating MDA-MB-231 cells in vitro [12][13][14] . Metabolic adaptations enable survival of cancer cells in an anchorage-independent condition 6,[15][16][17][18] . By stimulating glucose uptake, oncogenes restore redox and energy balance and prevent anoikis in breast cancer cells 18 .

Epidemiological studies indicate that metformin, a widely used type 2 diabetes drug, might reduce breast cancer risk and mortality in patients with type 2 diabetes. Metformin might protect against breast cancer indirectly by ameliorating systemic glucose homeostasis. Alternatively, it might target breast cancer cells directly. However, experiments using MDA-MB-231 cells, a standard in vitro breast cancer model, produced inconsistent results regarding effectiveness of metformin as a direct anti-cancer agent. Metformin treatments in cultured MDA-MB-231 cells are usually performed for 48–96 hours, but protocols describing renewal of cell culture medium during these prolonged treatments are rarely reported. We determined whether medium renewal protocol might alter sensitivity of MDA-MB-231 cells treated with metformin. Using the MTS assay, BrdU incorporation and Hoechst staining we found that treatment with metformin for 48–72 hours failed to suppress viability and proliferation of MDA-MB-231 cells if low-glucose (1 g/L) medium was renewed every 24 hours. Conversely, metformin suppressed their viability and proliferation if medium was not renewed. Without renewal glucose concentration in the medium was reduced to 0.1 g/L in 72 hours, which likely explains increased sensitivity to metformin under these conditions. We also examined whether 2-deoxy-D-glucose (2-DG) reduces resistance to metformin. In the presence of 2-DG metformin reduced viability and proliferation of MDA-MB-231 cells with or without medium renewal, thus demonstrating that 2-DG reduces their resistance to metformin. In sum, we show that medium renewal blocks anti-proliferative effects of metformin during prolonged treatments in low-glucose medium. Differences in medium renewal protocols during prolonged treatments might therefore lead to apparently inconsistent results as regards effectiveness of metformin as a direct anti-cancer agent. Finally, our results indicate that co-therapy with 2-DG and metformin might provide an effective strategy to overcome metformin resistance of breast cancer cells.

Pirkmajer et al. Ouabain Modulates Myocyte IL-6/STAT3 Signaling might represent a negative feedback in the IL-6/STAT3 pathway. Collectively, our results implicate a role for CTS and NKA in regulation of the IL-6 signaling and secretion in skeletal muscle.