Purpose Genome-wide DNA methylation analyses have identified hundreds of candidate DNA-hypermethylated genes in cancer. Comprehensive functional analyses provide an understanding of the biologic significance of this vast amount of DNA methylation data that may allow the determination of key epigenetic events associated with tumorigenesis. Experimental Design To study mechanisms of cysteine dioxygenase type 1 (CDO1) inactivation and its functional significance in breast cancer in a comprehensive manner, we screened for DNA methylation and gene mutations in primary breast cancers and analyzed growth, survival, and reactive oxygen species (ROS) production in breast cancer cells with restored CDO1 function in the context of anthracycline treatment. Results DNA methylation-associated silencing of CDO1 in breast cancer is frequent (60%), cancer specific, and correlates with disease progression and outcome. CDO1 function can alternatively be silenced by repressive chromatin, and we describe protein-damaging missense mutations in 7% of tumors without DNA methylation. Restoration of CDO1 function in breast cancer cells increases levels of ROS and leads to reduced viability and growth, as well as sensitization to anthracycline treatment. Priming with 5-azacytidine of breast cancer cells with epigenetically silenced CDO1 resulted in restored expression and increased sensitivity to anthracyclines. Conclusion We report that silencing of CDO1 is a critical epigenetic event that contributes to the survival of oxidative-stressed breast cancer cells through increased detoxification of ROS and thus leads to the resistance to ROS-generating chemotherapeutics including anthracyclines. Our study shows the importance of CDO1 inactivation in breast cancer and its clinical potential as a biomarker and therapeutic target to overcome resistance to anthracyclines.

The NTCP (Na⁺-taurocholate co-transporting protein)/SLC10A [solute carrier family 10 (Nav/bile acid co-transporter family)] 1 is tightly controlled to ensure hepatic bile salt uptake while preventing toxic bile salt accumulation. Many transport proteins require oligomerization for their activity and regulation. This is not yet established for bile salt transporters. The present study was conducted to elucidate the oligomeric state of NTCP. Chemical cross-linking revealed the presence of NTCP dimers in rat liver membranes and U2OS cells stably expressing NTCP. Co-immunoprecipitation of tagged NTCP proteins revealed a physical interaction between subunits. The C-terminus of NTCP was not required for subunit interaction, but was essential for exit from the ER (endoplasmic reticulum). NTCP without its C-terminus (NTCP Y307X) retained full-length wtNTCP (wild-type NTCP) in the ER in a dominant fashion, suggesting that dimerization occurs early in the secretory pathway. FRET (fluorescence resonance energy transfer) using fluorescently labelled subunits further demonstrated that dimerization persists at the plasma membrane. NTCP belongs to the SLC10A protein family which consists of seven members. NTCP co-localized in U2OS cells with SLC10A4 and SLC10A6, but not with SLC10A3, SLC10A5 or SLC10A7. SLC10A4 and SLC10A6 co-immunoprecipitated with NTCP, demonstrating that heteromeric complexes can be formed between SLC10A family members in vitro. Expression of SLC10A4 and NTCP Y307X resulted in a reduction of NTCP abundance at the plasma membrane and NTCP-mediated taurocholate uptake, whereas expression of SLC10A6 or NTCP E257N, an inactive mutant, did not affect NTCP function. In conclusion, NTCP adopts a dimeric structure in which individual subunits are functional. Bile salt uptake is influenced by heterodimerization when this impairs NTCP plasma membrane trafficking.

Bile acids are pivotal for the absorption of dietary lipids and vitamins and function as important signaling molecules in metabolism. Here, we describe a genetically encoded fluorescent bile acid sensor (BAS) that allows for spatiotemporal monitoring of bile acid transport in single living cells. Changes in concentration of multiple physiological and pathophysiological bile acid species were detected as robust changes in F€ orster resonance energy transfer (FRET) in a range of cell types. Specific subcellular targeting of the sensor demonstrated rapid influx of bile acids into the cytoplasm and nucleus, but no FRET changes were observed in the peroxisomes. Furthermore, expression of the liver fatty acid binding protein reduced the availability of bile acids in the nucleus. The sensor allows for single cell visualization of uptake and accumulation of conjugated bile acids, mediated by the Na 1 -taurocholate cotransporting protein (NTCP). In addition, cyprinol sulphate uptake, mediated by the putative zebrafish homologue of the apical sodium bile acid transporter, was visualized using a sensor based on the zebrafish farnesoid X receptor. The reversible nature of the sensor also enabled measurements of bile acid efflux in living cells, and expression of the organic solute transporter ab (OSTab) resulted in influx and efflux of conjugated chenodeoxycholic acid. Finally, combined visualization of bile acid uptake and fluorescent labeling of several NTCP variants indicated that the sensor can also be used to study the functional effect of patient mutations in genes affecting bile acid homeostasis. Conclusion: A genetically encoded fluorescent BAS was developed that allows intracellular imaging of bile acid homeostasis in single living cells in real time. (HEPATOLOGY 2013;57:740-752) B ile acids operate as signaling molecules with systemic endocrine functions that are very important for lipid, glucose, and energy homeostasis and are essential for intestinal absorption of dietary fat and fat-soluble vitamins.