Ogretmen, B. | Search bioRxiv

bioRxiv2023

Targeting LINC00152 activates cAMP/Ca2+/ferroptosis axis and overcomes tamoxifen resistance in ER+ breast cancer

Tamoxifen has been the mainstay therapy to treat early, locally advanced, and metastatic estrogen receptor-positive (ER+) breast cancer, constituting around 75% of all cases. However, emergence of resistance is common, necessitating the identification of novel therapeutic targets. Here, we demonstrated that long-noncoding RNA LINC00152 confers tamoxifen resistance via blocking tamoxifen-induced ferroptosis, an iron-mediated cell death. Mechanistically, inhibiting LINC00152 reduces the mRNA stability of phosphodiesterase 4D (PDE4D), leading to activation of cAMP/PKA/CREB axis and increased expression of TRPC1 Ca2+ channel. This causes cytosolic Ca2+ overload and generation of reactive oxygen species (ROS) that is, on one hand, accompanied by downregulation of FTH1, a member of the iron sequestration unit, thus increasing intracellular Fe2+ levels; and on the other hand, inhibition of the peroxidase activity upon reduced GPX4 and xCT levels. These ultimately induce lipid peroxidation and ferroptotic cell death in combination with tamoxifen. Overexpressing PDE4D rescues LINC00152 inhibition-mediated tamoxifen sensitization by de-activating the cAMP/Ca2+/ferroptosis axis. Importantly, high LINC00152 expression is significantly correlated with high PDE4D/low ferroptosis and worse survival in multiple cohorts of tamoxifen- or tamoxifen-containing endocrine therapy-treated ER+ breast cancer patients. Overall, we identified LINC00152 inhibition as a novel mechanism of ferroptosis induction and tamoxifen sensitization, thereby revealing LINC00152 and its effectors as actionable therapeutic targets to improve clinical outcome in refractory ER+ breast cancer.

cancer biology↗

bioRxiv2022

Ceramide synthase 6 (CerS6) promotes alcohol-induced fatty liver by promoting metabolic dysfunction and upregulating lipid droplet-associated proteins

ObjectiveAlcohol-associated liver disease (ALD) is the leading cause of liver-related mortality worldwide. Current strategies to manage ALD largely focus on advanced stage disease, however, metabolic changes such as glucose intolerance are apparent at the earliest stage of alcoholic steatosis and increase the risk of disease progression. Ceramides impair insulin signaling and accumulate in ALD, and metabolic pathways involving ceramide synthase 6 (CerS6) are perturbed in ALD during hepatic steatosis. In this study, we aimed to investigate the role of CerS6 in ALD development. MethodsC57BL/6 WT and CerS6 KO mice of both sexes were fed either a Lieber-DeCarli control (CON) or 15% ethanol (EtOH) diet for 6 weeks. In vivo metabolic tests including glucose and insulin tolerance tests (GTT and ITT) were performed. The mice were euthanized, and liver histology and lipid levels in serum and liver were measured. For in vitro studies, CerS6 was deleted in human hepatocytes and were incubated with EtOH and/or C16:0-ceramides. RNAseq analysis was performed in mice and in liver from patients with different stages of ALD and diseased controls. ResultsAfter six weeks on an EtOH diet, CerS6 KO mice had reduced body weight, food intake, and %fat mass compared to WT mice. Male (but not female) EtOH-fed KO mice showed significantly higher O2 consumption, CO2 production, respiratory exchange ratio, and energy expenditure (P<0.05 for all) during the dark period compared to EtOH-fed WT mice. In response to EtOH, WT mice developed mild hepatic steatosis, while steatosis was alleviated in KO mice as determined by H&E and ORO staining. KO mice showed significantly decreased long-chain ceramide species, especially C16:0 ceramides, in the serum and liver tissues compared to WT mice. CerS6 deletion decreased serum TG and NEFA only in male not female mice. CerS6 deletion improved glucose tolerance and insulin resistance in EtOH-fed mice of both sexes. RNAseq analysis revealed that 74 genes are significantly upregulated and 66 genes are downregulated by CerS6 deletion in EtOH-fed male mice, with key network pathways including TG biosynthetic process, positive regulation of lipid localization, and fat cell differentiation. Similar to RNAseq results, absence of CerS6 significantly decreased mRNA expression of lipid droplet associated proteins in EtOH-fed mice. In vitro, EtOH stimulation significantly increased PLIN2 protein expression in VL-17A cells while CerS6 deletion inhibited EtOH-mediated PLIN2 upregulation. C16:0-ceramide treatment significantly increased PLIN2 protein expression compared to CON. Importantly, progression of ALD in humans was associated with increased CerS6 hepatic expression. ConclusionsOur findings demonstrate that CerS6 deletion attenuates EtOH-induced weight gain and hepatic steatosis and improves glucose homeostasis in mice fed an EtOH diet. Notably, we unveil that CerS6 plays a major role as a regulator of lipid droplet biogenesis in alcoholic intra-hepatic lipid droplet formation. Together, our data suggest that CerS6 may be targeted for treatment for early stage ALD.

pathology↗