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Background Doxorubicin (Dox) is a widely used chemotherapy, but its effectiveness is limited by dose-dependent side effects. Although lower Dox doses reduce this risk, studies have reported higher recurrence of local disease with no improvement in survival rate in patients receiving low doses of Dox. To effectively mitigate this, a better understanding of the adverse effects of suboptimal Dox doses is needed. Methods Effects of sublethal dose of Dox on phenotypic changes were assessed with light and confocal microscopy. Migratory and invasive behavior were assessed by wound healing and transwell migration assays. MTT and LDH release assays were used to analyze cell growth and cytotoxicity. Flow cytometry was employed to detect cell surface markers of cancer stem cell population. Expression and activity of matrix metalloproteinases were probed with qRT-PCR and zymogen assay. To identify pathways affected by sublethal dose of Dox, exploratory RNAseq was performed and results were verified by qRT-PCR in multiple cell lines (MCF7, ZR75-1 and U-2OS). Regulation of Src Family kinases (SFK) by key players in DNA damage response was assessed by siRNA knockdown along with western blot and qRT-PCR. Dasatinib and siRNA for Fyn and Yes was employed to inhibit SFKs and verify their role in increased migration and invasion in MCF7 cells treated with sublethal doses of Dox. Results The results show that sublethal Dox treatment leads to increased migration and invasion in otherwise non-invasive MCF7 breast cancer cells. Mechanistically, these effects were independent of the epithelial mesenchymal transition, were not due to increased cancer stem cell population, and were not observed with other chemotherapies. Instead, sublethal Dox induces expression of multiple SFK—including Fyn, Yes, and Src—partly in a p53 and ATR-dependent manner. These effects were validated in multiple cell lines. Functionally, inhibiting SFKs with Dasatinib and specific downregulation of Fyn suppressed Dox-induced migration and invasion of MCF7 cells. Conclusions Overall, this study demonstrates that sublethal doses of Dox activate a pro-invasive, pro-migration program in cancer cells. Furthermore, by identifying SFKs as key mediators of these effects, our results define a potential therapeutic strategy to mitigate local invasion through co-treatment with Dasatinib.

It is now appreciated that sphingolipids constitute a rich class of bioactive molecules that include ceramide, sphingosine, and sphingosine 1‐phosphate whose formation is controlled by a network of highly regulated enzymes (Hannun & Obeid, 2008 ). Notably, several stress stimuli induce the production of ceramide, which, as a single entity, has been traditionally associated with apoptotic and growth suppressive functions. However, recent data clearly suggest that this simplistic formulation is no longer tenable. Graphical Abstract Hannun and Newcomb comment on the paper published in this issue providing evidence of cross talk between platelets and tumor cells via acid sphingomyelinase‐mediated trans‐cellular signaling, required for lung seeding of melanoma cells.

Introduction. Antiretroviral therapy (ART) has reduced mortality and improved life expectancy among HIV patients but does not provide a cure. Patients must remain on lifelong medications and deal with drug resistance and side effects. This underscores the need for HIV cure research. However, participation in HIV cure research has risks without guaranteed benefits. We determined what HIV healthcare providers know about HIV cure research trials, the risks involved, and what kind of cure interventions they are likely to recommend for their patients. Methods. We conducted in-depth qualitative interviews with 39 HIV care providers consisting of 12 physicians, 8 counsellors, 14 nurses, 2 pharmacists, 2 laboratory scientists, and 1 community advocate from three hospitals. Interviews were transcribed verbatim and coded, and thematic analysis was performed independently by two investigators. Results. Participants were happy about the success of current treatments and hopeful that an HIV cure will be found in the near future, just as ART was discovered through research. They described cure as total eradication of the virus from the body and inability to test positive for HIV or transmit the virus. In terms of risk tolerance, respondents would recommend to their patients’ studies with mild to moderate risks like what patients on antiretroviral therapy experience. Participants were reluctant to recommend treatment interruption to patients as part of a cure study and wished trials could be performed without stopping treatment. Healthcare providers categorically rejected death or permanent disability as an acceptable risk. The possibility of finding a cure that will benefit the individual or future generations was strong motivations for providers to recommend cure trials to their patients, as was transparency and adequate information on proposed trials. Overall, the participants were not actively seeking knowledge on cure research and lacked information on the various cure modalities under investigation. Conclusion. While hopeful for an HIV cure, healthcare providers in Ghana expect a cure to be definitive and pose minimal risk to their patients.

arising from N. C. Derecki et al. Nature484, 105–109 (2012); doi:10.1038/nature10907 Rett syndrome is a severe neurodevelopmental disorder caused by mutations in the X chromosomal gene MECP2 (ref. 1 ), and its treatment so far is symptomatic. Mecp2 disruption in mice phenocopies major features of the syndrome 2 that can be reversed after Mecp2 re-expression 3 . Recently, Derecki et al. 4 reported that transplantation of wild-type bone marrow into lethally irradiated Mecp2 -null ( Mecp2 tm1.1Jae/y ) mice prevented neurological decline and early death by restoring microglial phagocytic activity against apoptotic targets 4 , and clinical trials of bone marrow transplantation (BMT) for patients with Rett syndrome have thus been initiated 5 . We aimed to replicate and extend the BMT experiments in three different Rett syndrome mouse models, but found that despite robust microglial engraftment, BMT from wild-type donors did not prevent early death or ameliorate neurological deficits. Furthermore, early and specific Mecp2 genetic expression in microglia did not rescue Mecp2 -deficient mice.

Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 ( ACTA2 ) and Alpha-1 Type I Collagen ( COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.

Nature 521, E1–E4 (2015); doi:10.1038/nature14444 In this Brief Communication Arising, the first name of author Sébastien Vingeau was misspelled ‘Sebastian’. In addition, the labels (‘WT→KO’ and ‘KO→WT’) of the two bottom panels in Extended Data Figure 1b were swapped. Both errors have been corrected online.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in the X chromosomal gene Methyl-CpG-binding Protein 2 (MECP2) (1). RTT treatment so far is symptomatic. Mecp2 disruption in mice phenocopies major features of the syndrome (2) that can be reversed upon re-expression of Mecp2 (3. It has recently been reported that transplantation of wild type (WT) bone marrow (BMT) into lethally irradiated Mecp2tm1.1Jae/y mice prevented neurologic decline and early death by restoring microglial phagocytic activity against apoptotic targets (4). Based on this report, clinical trials of BMT for patients with RTT have been initiated (5). We aimed to replicate and extend the BMT experiments in three different RTT mouse models but found that despite robust microglial engraftment, BMT from WT donors did not rescue early death or ameliorate neurologic deficits. Furthermore, early and specific genetic expression of Mecp2 in microglia did not rescue Mecp2-deficient mice. In conclusion our experiments do not support BMT as therapy for RTT.

Acid Sphingomyelianse (ASM) is a key regulatory enzyme that produces the bioactive lipid ceramide, which is converted into other sphingolipids by enzymes such as Ceramide Kinase (CERK). Previous literature has implicated ASM and CERK in inflammatory signaling, but the specific role of ASM and CERK as mediators of inflammatory signaling in breast cancer has remained elusive. In this work, we delineated the role of ASM in p38 activation and subsequent IL-6 production, as well as the role of CERK as a downstream effector of activated ASM. Since previous literature has shown that TNF-α induced dramatic alterations in the sphingolipid profile of cells, sphingolipidomic analysis was carried out on TNF-α treated MCF7 cells. The data revealed that TNF-α induced robust production of ceramide-1-phosphate (C-1-P) that accompanied an increase in CCL5. Given that previous literature showed CCL5 production is dependent on ASM, this data suggested that CERK may be acting downstream of ASM to produce C-1-P and induce CCL5. Indeed, siRNA ablation of CERK decreased CCL5 production. Furthermore, previous literature implicated C-1-P in Golgi localization of cPLA2. To define a role for ASM as an upstream modulator of CERK activity, the requirement for ASM in cPLA2 localization was tested. Ablation of ASM blocked cPLA2 localization to the Golgi network, suggesting ASM is acting upstream of CERK. Since ASM appeared to have Golgi specific functions, ASM mutants that accumulate in the Golgi network were tested for the ability to induce CCL5. We found that Golgi targeted ASM can induce CCL5 production. Taken together, these results demonstrate a role for CERK and C-1-P in CCL5 production and a role for ASM in modulation of CERK function. These results highlight the previously unappreciated role of C-1-P in CCL5 production and present new opportunities for targeting sphingolipid metabolism in treatment of breast cancers.