Measurements of sphingolipid metabolism are most accurately performed by LC-MS. However, this technique is expensive, not widely accessible, and without the use of specific probes, it does not provide insight into metabolic flux through the pathway. Employing the fluorescent ceramide analogue NBD-C6-ceramide as a tracer in intact cells, we developed a comprehensive HPLC-based method that simultaneously measures the main nodes of ceramide metabolism in the Golgi. Hence, by quantifying the conversion of NBD-C6-ceramide to NBD-C6-sphingomyelin, NBD-C6-hexosylceramides, and NBD-C6-ceramide-1-phosphate (NBD-C1P), the activities of Golgi resident enzymes sphingomyelin synthase 1, glucosylceramide synthase, and ceramide kinase (CERK) could be measured simultaneously. Importantly, the detection of NBD-C1P allowed us to quantify CERK activity in cells, a usually difficult task. By applying this method, we evaluated the specificity of commonly used sphingolipid inhibitors and discovered that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, which targets glucosylceramide synthase, and fenretinide (4HPR), an inhibitor for dihydroceramide desaturase, also suppress CERK activity. This study demonstrates the benefit of an expanded analysis of ceramide metabolism in the Golgi, and it provides a qualitative and easy-to-implement method.

Dysregulated sphingolipid metabolism contributes to ER+ breast cancer progression and therapeutic response, whereas its underlying mechanism and contribution to tamoxifen resistance (TAMR) is unknown. Here, we establish sphingolipid metabolic enzyme CERK as a regulator of TAMR in breast cancer. Multi-omics analysis reveals an elevated CERK driven sphingolipid metabolic reprogramming in TAMR cells, while high CERK expression associates with worse patient prognosis in ER+ breast cancer. CERK overexpression confers tamoxifen resistance and promotes tumorigenicity in ER+ breast cancer cells. Knocking out CERK inhibits the orthotopic breast tumor growth of TAMR cells while rescuing their tamoxifen sensitivity. Mechanistically, the elevated EHF expression transcriptionally up-regulates CERK expression to prohibit tamoxifen-induced sphingolipid ceramide accumulation, which then inhibits tamoxifen-mediated repression on PI3K/AKT dependent cell proliferation and its driven p53/caspase-3 mediated apoptosis in TAMR cells. This work provides insight into the regulation of sphingolipid metabolism in tamoxifen resistance and identifies a potential therapeutic target for this disease.

BACKGROUND: Clinical management of triple-negative breast cancer (TNBC) patients remain challenging because of the development of chemo-resistance. Identification of biomarkers for risk stratification of chemo-resistance and therapeutic decision-making to overcome such resistance is thus necessary.
METHODS: Retrospective analysis was performed to identify potential stratification biomarkers. The levels of ceramide kinase (CERK) was determined in breast cancer patients. The roles of CERK and its downstream signaling pathways were analysed using cellular and biochemical assays.
RESULTS: CERK upregulation was identified as a biomarker for chemotherapeutic response in TNBC. A > 2-fold change in CERK (from tumor)/CERK (from normal counterpart) was significantly associated with chemo-resistance (OR = 2.66, 95% CI 1.18-7.34), P = 0.04. CERK overexpression was sufficient to promote TNBC growth and migration, and confer chemo-resistance in TNBC cell lines, although this resistance could be overcome via CERK inhibition. Mechanistic studies suggest that CERK mediates intrinsic resistance and inferior response to chemotherapy in TNBC by regulating multiple oncogenic pathways such as Ras/ERK, PI3K/Akt/mTOR, and RhoA.
CONCLUSIONS: Our work provides an explanation for the heterogeneity of chemo-response across TNBC patients and demonstrates that CERK inhibition offers a therapeutic strategy to overcome treatment resistance.

Aging skeletal muscle shows perturbations in metabolic functions. MicroRNAs have been shown to play a critical role in aging and metabolic functions of skeletal muscle. MicroRNA-34a (miR-34a) is implicated in the brain and cardiac aging, however, its role in aging muscle is unclear. We analyzed levels of miR-34a, ceramide kinase (CERK) and other insulin signaling molecules in skeletal muscle from old mice. In addition to in vivo model, levels of these molecules were also analyzed in myoblast derived from insulin resistant (IR) humans and C2C12 myoblasts overexpressing mir-34a. Our results show that miR-34a is elevated in the muscles of 2-year-old mice and in the myoblasts of IR humans. Overexpression of miR-34a in C2C12 myoblasts leads to alterations in the insulin signaling pathway, which were rescued by its antagonism. Our analyses revealed that miR-34a targets CERK resulting in ceramide accumulation, activation of PP2A and the pJNK pathway in muscle and C2C12 myoblasts. Also, myostatin (Mstn) levels were increased in 2-year-old mouse muscle and Mstn treatment upregulated miR-34a in C2C12 myoblasts. In addition, miR-34a expression and ceramide levels did not increase during aging in Mstn-/- mice muscle. In summary, we, therefore, propose that Mstn levels increase in aging muscle and upregulate miR-34a, which inhibits CERK resulting in increased ceramide levels. This ceramide accumulation activates PP2A and pJNK causing hypophosphorylation of AKT and hyperphosphorylation of IRS1 (Ser307), respectively, impairing insulin signaling pathway and eventually inhibiting the sarcolemma localization of GLUT4. These changes would result in reduced glucose uptake and insulin resistance. This study is the first to explain the phenomenon of ceramide accrual and impairment of insulin signaling pathway in aging muscle through a miR-34a based mechanism. In conclusion, our results suggest that Mstn and miR-34a antagonism can help ameliorate ceramide accumulation and loss of insulin sensitivity in aging skeletal muscle.

The receptor like kinases (RLKs) belong to the RLK/Pelle superfamily, one of the largest gene families in plants. RLKs play an important role in plant development, as well as in response to biotic and abiotic stresses. The lysine motif receptor like kinases (LysM-RLKs) are a subfamily of RLKs containing at least one lysine motif (LysM) that are involved in the perception of elicitors or pathogen-associated molecular patterns (PAMPs). In the present study, 77 putative RLKs genes and three receptor like proteins were identified in potato (Solanum tuberosum) genome, following a genome-wide search. The 77 potato RLK proteins are classified into two major phylogenetic groups based on their kinase domain amino acid sequence similarities. Out of 77 RLKs, 10 proteins had at least one LysM. Among them three RLP proteins were found in potato genome with either 2 or three tandem LysM but these lacked a cytoplasmic kinase domain. Expression analyses of a potato LysM-RLKs (StLysM-RLK05) was carried out by a Real time RT-PCR, following inoculation of potato leaves and immature tubers with late blight and common scab pathogens, respectively. The expression was significantly higher in resistant than in susceptible following S. scabies inoculation. The StLysM-RLK05 sequence was verified and it was polymorphic in scab susceptible cultivar. The present study provides an overview of the StLysM-RLKs gene family in potato genome. This information is helpful for future functional analysis of such an important protein family, in Solanaceae species.