Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment.

Bioactive sphingolipids are modulators of immune processes and their metabolism is often dysregulated in ulcerative colitis, a major category of inflammatory bowel disease (IBD). While multiple axes of sphingolipid metabolism have been investigated to delineate mechanisms regulating ulcerative colitis, the role of acid ceramidase (AC) in intestinal inflammation is yet to be characterized. Here we demonstrate that AC expression is elevated selectively in the inflammatory infiltrate in human and murine colitis. To probe for mechanistic insight into how AC up-regulation can impact intestinal inflammation, we investigated the selective loss of AC expression in the myeloid population. Using a model of intestinal epithelial injury, we demonstrate that myeloid AC conditional knockout mice exhibit impairment of neutrophil recruitment to the colon mucosa as a result of defective cytokine and chemokine production. Furthermore, the loss of myeloid AC protects from tumor incidence in colitis-associated cancer (CAC) and inhibits the expansion of neutrophils and granulocytic myeloid-derived suppressor cells in the tumor microenvironment. Collectively, our results demonstrate a tissue-specific role for AC in regulating neutrophilic inflammation and cytokine production. We demonstrate novel mechanisms of how granulocytes are recruited to the colon that may have therapeutic potential in intestinal inflammation, IBD, and CAC.-Espaillat, M. P., Snider, A. J., Qiu, Z., Channer, B., Coant, N., Schuchman, E. H., Kew, R. R., Sheridan, B. S., Hannun, Y. A., Obeid, L. M. Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment.

Acid ceramidase (AC)--a key enzyme of sphingolipid metabolism--correlates with better prognosis in epithelial ovarian cancer.

Acid ceramidase (AC), a key enzyme of sphingolipid metabolism, seems to play an important role in cancer progression. The objective of this study was to explore the expression of AC in ovarian cancer and its impact on prognosis. Expression analysis of AC in n=112 ovarian cancer patients was performed by immunohistochemical analysis of primary paraffin-embedded tumor samples. The results were scored on the basis of the staining intensity and percentage of positive tumor cells, resulting in an immunoreactive score from 0 to 12. These results were correlated to clinical and pathologic characteristics and survival. AC expression correlated significantly only with FIGO stage (0.047). In serous carcinoma, low level of AC was independently associated with reduced progression-free survival and overall survival of 12.0 mo [95% confidence interval (CI), 5.78-18.23] versus 18.1 mo (95% CI, 11.61-24.59; P=0.008) and 35.7 mo (95% CI, 22.24-47.16) versus 58.7 mo (95% CI, 36.48-80.91; P=0.032), respectively. In multivariate analysis, AC presents as an independent prognostic factor for progression-free survival (hazard ratio 1.88; 95% CI, 1.13-3.11; P=0.015). AC is a prognostic factor in epithelial ovarian cancer. Low AC expression can be associated with tumor progression in carcinoma of the ovaries. These results are in contrast to the concept of AC as a promoter for cancer progression. Nevertheless, they are supported by the lately discovered tumor-suppressing function of sphingosine, the enzymatic product of AC.

Acid ceramidase expression modulates the sensitivity of A375 melanoma cells to dacarbazine.

Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma.

Genistein stimulates MCF-7 breast cancer cell growth by inducing acid ceramidase (ASAH1) gene expression.

Sphingolipid metabolites, such as ceramide (Cer), sphingosine (SPH), and sphingosine 1-phosphate (S1P), contribute to multiple aspects of carcinogenesis including cell proliferation, migration, angiogenesis, and tumor resistance. The cellular balance between Cer and S1P levels, for example, is an important determinant of cell fate, with the former inducing apoptosis and the later mitogenesis. Acid ceramidase (ASAH1) plays a pivotal role in regulating the intracellular concentration of these two metabolites by hydrolyzing Cer into SPH, which is rapidly phosphorylated to form S1P. Genistein is a phytoestrogen isoflavone that exerts agonist and antagonist effects on the proliferation of estrogen-dependent MCF-7 cells in a dose-dependent manner, primarily as a ligand for estrogen receptors. Genistein can also activate signaling through GPR30, a G-protein-coupled cell surface receptor. Based on the relationship between bioactive sphingolipids and tumorigenesis, we sought to determine the effect of genistein on ASAH1 transcription in MCF-7 breast cancer cells. We show herein that nanomolar concentrations of genistein induce ASAH1 transcription through a GPR30-dependent, pertussis toxin-sensitive pathway that requires the activation of c-Src and extracellular signal regulated kinase 1/2 (ERK1/2). Activation of this pathway promotes histone acetylation and recruitment of phospho-estrogen receptor α and specificity protein-1 to the ASAH1 promoter, ultimately culminating in increased ceramidase activity. Finally, we show that genistein stimulates cyclin B2 expression and cell proliferation in an ASAH1-dependent manner. Collectively, these data identify a mechanism through which genistein promotes sphingolipid metabolism and support a role for ASAH1 in breast cancer cell growth.

Effect of di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate on in vitro developmental competence of bovine oocytes.

In the last decade, potential exposure of humans and animals to industrial chemicals and pesticides has been a growing concern. In the present study, di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) were used to model the effects of endocrine-disrupting compounds and their risk in relation to early embryonic losses. Exposure of cumulus oocyte complexes during maturation to 50 µM MEHP reduced the proportion of oocytes that underwent nuclear maturation (p < 0.05) and increased the proportion of apoptotic oocytes (p < 0.05). Furthermore, phthalates reduced cleavage rate in the MEHP-treated group (p < 0.05) and the proportion of embryos developing to the blastocyst stage in both DEHP- and MEHP-treated groups (p < 0.05). The total cell count for blastocysts developing from MEHP-treated oocytes was lower than in controls (p < 0.05). Exposure of oocytes to MEHP during maturation reduced (p < 0.05) the expression of ASAH1 (an anti-apoptotic factor), CCNA2 (involved in cell cycle control), and POU5F1 (responsible for pluripotency) in matured oocytes. Furthermore, the reduced mRNA expression of POU5F1 and ASAH1 lasted into two-cell stage embryos (p < 0.05). Phthalate-induced alterations in POU5F1, ASAH1, and CCNA2 expression might explain in part the reduced developmental competence of MEHP-treated oocytes.

Pediatric ependymoma: GNAO1, ASAH1, IMMT and IPO7 protein expression and 5-year prognosis correlation.

OBJECTIVE: The aim of this work was to evaluate a pediatric ependymoma protein expression that may be useful as a molecular biomarker candidate for prognosis, correlated with clinical features such as age, gender, histopathological grade, ependymal tumor recurrence and patient survival. PATIENTS AND METHODS: Immunohistochemistry assays were performed for GNAO1, ASAH1, IMMT, IPO7, Cyclin D1, P53 and Ki-67 proteins. Kaplan-Meier and Cox analysis were performed for age, gender, histopathological grade, relapse and survival correlation. RESULTS: We found that three proteins correlate with histopathological grade and relapse; two proteins correlate with survival; one protein does not correlate with any clinical feature. CONCLUSION: Our results suggest that, out of the proteins analyzed, five may be considered suitable prognostic biomarkers and one may be considered a predictive biomarker for response to treatment of pediatric ependymoma.

Acid ceramidase confers radioresistance to glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common primary, intracranial malignancy of the central nervous system. The standard treatment protocol, which involves surgical resection, and concurrent radiation with adjuvant temozolomide (TMZ), still imparts a grim prognosis. Ultimately, all GBMs exhibit recurrence or progression, developing resistance to standard treatment. This study demonstrates that GBMs acquire resistance to radiation via upregulation of acid ceramidase (ASAH1) and sphingosine­1-phosphate (Sph-1P). Moreover, inhibition of ASAH1 and Sph-1P, either with humanized monoclonal antibodies, small molecule drugs (i.e. carmofur), or a combination of both, led to suppression of GBM cell growth. These results suggest that ASAH1 and Sph-1P may be excellent targets for the treatment of new GBMs and recurrent GBMs, especially since the latter overexpresses ASAH1.

The ART of Lowering Ceramides.

Ceramides are lipid metabolites implicated in the metabolic dysregulation that accompanies dyslipidaemia and obesity. Using a genetic mouse model to acutely degrade ceramides in adipose tissue or the liver (i.e., by conditionally expressing acid ceramidase), in this issue of Cell MetabolismXia et al. (2015) identify roles for these molecules in insulin resistance, steatohepatitis, and interorgan communication.

Targeted Induction of Ceramide Degradation Leads to Improved Systemic Metabolism and Reduced Hepatic Steatosis.

Sphingolipids have garnered attention for their role in insulin resistance and lipotoxic cell death. We have developed transgenic mice inducibly expressing acid ceramidase that display a reduction in ceramides in adult mouse tissues. Hepatic overexpression of acid ceramidase prevents hepatic steatosis and prompts improvements in insulin action in liver and adipose tissue upon exposure to high-fat diet. Conversely, overexpression of acid ceramidase within adipose tissue also prevents hepatic steatosis and systemic insulin resistance. Induction of ceramidase activity in either tissue promotes a lowering of hepatic ceramides and reduced activation of the ceramide-activated protein kinase C isoform PKCζ, though the induction of ceramidase activity in the adipocyte prompts more rapid resolution of hepatic steatosis than overexpression of the enzyme directly in the liver. Collectively, our observations suggest the existence of a rapidly acting "cross-talk" between liver and adipose tissue sphingolipids, critically regulating glucose metabolism and hepatic lipid uptake.

Acid ceramidase is associated with an improved prognosis in both DCIS and invasive breast cancer.

Acid ceramidase (ASAH1) a key enzyme of sphingolipid metabolism converting pro-apoptotic ceramide to sphingosine has been shown to be overexpressed in various cancers. We previously demonstrated higher expression of ASAH1 in ER positive compared to ER negative breast cancer. In the current study we performed subtype specific analyses of ASAH1 gene expression in invasive and non invasive breast cancer. We show that expression of ASAH1 is mainly associated with luminal A - like cancers which are known to have the best prognosis of all breast cancer subtypes. Moreover tumors with high ASAH1 expression among the other subtypes are also characterized by an improved prognosis. The good prognosis of tumors with high ASAH1 is independent of the type of adjuvant treatment in breast cancer and is also detected in non small cell lung cancer patients. Moreover, even in pre-invasive DCIS of the breast ASAH1 is associated with a luminal phenotype and a reduced frequency of recurrences. Thus, high ASAH1 expression is generally associated with an improved prognosis in invasive breast cancer independent of adjuvant treatment and could also be valuable as prognostic factor for pre-invasive DCIS.

Fas ligand gene transfer effectively induces apoptosis in head and neck cancer cells.

CONCLUSION: Fas ligand (FasL) gene therapy may provide a new efficient therapeutic model for head and neck squamous cell cancer (HNSCC). Acid ceramidase (AC) may not play an important role in the sensitivity of HNSCC cell lines to Fas-induced apoptosis. OBJECTIVES: The aims of this study were to investigate the efficacy of FasL gene therapy for HNSCC in vitro and to determine whether the expression of AC in different kinds of HNSCC cell lines is related to the sensitivity of HNSCC cell lines to Fas-mediated apoptotic induction. METHODS: Three HNSCC cell lines (Hep-2, MMSI, and SCCVII) were transfected with pEGFP-FasL, a plasmid containing a modified human FasL gene fused to enhanced green fluorescent protein (GFP). pEGFP-C1, a plasmid containing the GFP gene alone, was used as a control. Cell death was observed by fluorescence imaging and quantified using a tetrazolium-based (MTS) assay. SCCVII cells were analyzed by flow cytometry to determine the presence of apoptotic induction. Hep-2 and MMSI cells were evaluated by quantitative real-time PCR to evaluate the expression of AC. RESULTS: Transfection of pEGFP-FasL plasmid was shown to be able to induce cell death, the sensitivity of Fas-mediated apoptosis in HNSCC was different, and the level of AC did not correlate with the sensitivity of HNSCC cells to Fas-induced apoptosis.

IRF8 regulates acid ceramidase expression to mediate apoptosis and suppresses myelogeneous leukemia.

IFN regulatory factor 8 (IRF8) is a key transcription factor for myeloid cell differentiation and its expression is frequently lost in hematopoietic cells of human myeloid leukemia patients. IRF8-deficient mice exhibit uncontrolled clonal expansion of undifferentiated myeloid cells that can progress to a fatal blast crisis, thereby resembling human chronic myelogeneous leukemia (CML). Therefore, IRF8 is a myeloid leukemia suppressor. Whereas the understanding of IRF8 function in CML has recently improved, the molecular mechanisms underlying IRF8 function in CML are still largely unknown. In this study, we identified acid ceramidase (A-CDase) as a general transcription target of IRF8. We demonstrated that IRF8 expression is regulated by IRF8 promoter DNA methylation in myeloid leukemia cells. Restoration of IRF8 expression repressed A-CDase expression, resulting in C16 ceramide accumulation and increased sensitivity of CML cells to FasL-induced apoptosis. In myeloid cells derived from IRF8-deficient mice, A-CDase protein level was dramatically increased. Furthermore, we demonstrated that IRF8 directly binds to the A-CDase promoter. At the functional level, inhibition of A-CDase activity, silencing A-CDase expression, or application of exogenous C16 ceramide sensitized CML cells to FasL-induced apoptosis, whereas overexpression of A-CDase decreased CML cells' sensitivity to FasL-induced apoptosis. Consequently, restoration of IRF8 expression suppressed CML development in vivo at least partially through a Fas-dependent mechanism. In summary, our findings determine the mechanism of IRF8 downregulation in CML cells and they determine a primary pathway of resistance to Fas-mediated apoptosis and disease progression.

Acid ceramidase upregulation in prostate cancer: role in tumor development and implications for therapy.

Bioactive sphingolipids, such as ceramide, sphingosine and sphingosine-1-phosphate are known bio-effector molecules which play important roles in various aspects of cancer biology including cell proliferation, growth arrest, apoptosis, metastasis, senescence and inflammation. Therefore, enzymes involved in ceramide metabolism are gaining recognition as being critical regulators of cancer cell growth and/or survival. We previously observed that the ceramide metabolizing enzyme, acid ceramidase (AC) is upregulated in tumor tissues. Studies have now concluded that this creates a dysfunctional ceramide pathway, which is responsible for tumor progression and resistance to chemotherapy and radiation. This suggests that development of small-molecule drugs that inhibit AC enzyme activity is a promising approach for improving standard cancer therapy and patient's clinical outcomes.