Inhibition of acid ceramidase regulates MHC class II antigen presentation and suppression of autoimmune arthritis.

The bioactive sphingolipid ceramide affects immune responses although its effect on antigen (Ag) processing and delivery by HLA class II to CD4+T-cells remains unclear. Therefore, we examined the actions of a novel cell-permeable acid ceramidase (AC) inhibitor [(1R,2R) N myristoylamino-(4'-nitrophenyl)-propandiol-1,3] on antigen presentation and inflammatory cytokine production by Ag-presenting cells (APCs) such as B-cells, macrophages, and dendritic cells. We found that AC inhibition in APCs perturbed Ag-processing and presentation via HLA-DR4 (MHC class II) proteins as measured by coculture assay and T-cell production of IL-2. Mass spectral analyses showed that B13 treatment significantly raised levels of four types of ceramides in human B-cells. B13 treatment did not alter Ag internalization and class II protein expression, but significantly inhibited lysosomal cysteinyl cathepsins (B, S and L) and thiol-reductase (GILT), HLA class II Ag-processing, and generation of functional class II-peptide complexes. Ex vivo Ag presentation assays showed that inhibition of AC impaired primary and recall CD4+T-cell responses and cytokine production in response against type II collagen. Further, B13 delayed onset and reduced severity of inflamed joints and cytokine production in the collagen-induced arthritis mouse model in vivo. These findings suggest that inhibition of AC in APCs may dysregulate endolysosomal proteases and HLA class II-associated self-antigen presentation to CD4+T-cells, attenuating inflammatory cytokine production and suppressing host autoimmune responses.

Tamoxifen is a candidate first-in-class inhibitor of acid ceramidase that reduces amitotic division in polyploid giant cancer cells-Unrecognized players in tumorigenesis.

Polyploid giant cancer cells (PGCC) represent a poorly understood, small subpopulation of tumor cells that are increasingly being recognized for their critical role in therapy resistance, metastasis, and cancer recurrence. PGCC have the potential to generate progeny through primitive or cleavage-like division, which allows them to evade antimitotic insults. We recently demonstrated that the sphingolipid enzyme acid ceramidase (ASAH1) is required for this process. Since specific ASAH1 inhibitors are not clinically available, we investigated whether tamoxifen, which interferes with ASAH1 function via off-target effects, has a potential clinical benefit independent of estrogen signaling. Our results show that tamoxifen inhibits generation of PGCC offspring in prostate cancer, glioblastoma, and melanoma cells. Analysis of two state-level cancer registries revealed that tamoxifen improves survival outcomes for second, nonbreast cancers that develop in women with early stage breast cancer. Our results suggest that tamoxifen may have a clinical benefit in a variety of cancers that is independent of estrogen signaling and could be due to its inhibition of acid ceramidase. Thus the distinct application of tamoxifen as potentially a first-in-class therapeutic that inhibits the generation of PGCC offspring should be considered in future clinical trials.

Genetic and pharmacological inhibition of acid ceramidase prevents asymmetric cell division by neosis.

Radiation treatment failure or relapse after initial response to chemotherapy presents significant clinical challenges in cancer patients. Escape from initial courses of treatment can involve reactivation of embryonic developmental stages, with the formation of polynuclear giant cancer cells (PGCCs). This strategy of dedifferentiation can insulate cancer cells from a variety of treatments and allows a residual subpopulation to reestablish tumors after treatment. Using radiation or docetaxel chemotherapy, we generated PGCCs from prostate cancer cells. Here, we show that expression of acid ceramidase (ASAH1), an enzyme in the sphingolipid pathway linked to therapy resistance and poor outcomes, is elevated in PGCCs. Targeting ASAH1 with shRNA or treatment with the ASAH1 inhibitor, LCL-521, did not impair the formation of PGCCs, but prevented the formation of PGCC progeny that arise through an asymmetric cell division called neosis. Similar results were obtained in lung cancer cells that had been exposed to radiation or cisplatin chemotherapy as stressors. In summary, our data suggest that endoreplication occurs independent of ASAH1 while neosis is ASAH1-dependent in both prostate and lung cancer cells. Because ASAH1 knockout is embryonic lethal but not deleterious to adult animals, targeting this enzyme has the potential to be highly specific to cells undergoing the dedifferentiation process to escape cancer treatments. Pharmacological inhibition of ASAH1 is a potentially powerful strategy to eliminate cells that could otherwise serve as seed populations for recurrence.

Interdiction of Sphingolipid Metabolism Revisited: Focus on Prostate Cancer.

Sphingolipid metabolism is known to play a role in cell death, survival, and therapy resistance in cancer. Sphingolipids, particularly dihydroceramide and ceramide, are associated with antiproliferative or cell death responses, respectively, and are central to effective cancer therapy. Within the last decade, strides have been made in elucidating many intricacies of sphingolipid metabolism. New information has emerged on the mechanisms by which sphingolipid metabolism is dysregulated during malignancy and how cancer cells survive and/or escape therapeutic interventions. This chapter focuses on three main themes: (1) sphingolipid enzymes that are dysregulated in cancer, particularly in prostate cancer; (2) inhibitors of sphingolipid metabolism that antagonize prosurvival responses; and (3) sphingolipid-driven escape mechanisms that allow cancer cells to evade therapies. We explore clinical and preclinical approaches to interdict sphingolipid metabolism and provide a rationale for combining strategies to drive the generation of antiproliferative ceramides with prevention of ceramide clearance.

Blocking Myristoylation of Src Inhibits Its Kinase Activity and Suppresses Prostate Cancer Progression.

Protein N-myristoylation enables localization to membranes and helps maintain protein conformation and function. N-myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N-myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR.

Acid Ceramidase Deficiency is characterized by a unique plasma cytokine and ceramide profile that is altered by therapy.

Acid Ceramidase Deficiency (Farber disease, FD) is an ultra-rare Lysosomal Storage Disorder that is poorly understood and often misdiagnosed as Juvenile Idiopathic Arthritis (JIA). Hallmarks of FD are accumulation of ceramides, widespread macrophage infiltration, splenomegaly, and lymphocytosis. The cytokines involved in this abnormal hematopoietic state are unknown. There are dozens of ceramide species and derivatives, but the specific ones that accumulate in FD have not been investigated. We used a multiplex assay to analyze cytokines and mass spectrometry to analyze ceramides in plasma from patients and mice with FD, controls, Farber patients treated by hematopoietic stem cell transplantation (HSCT), JIA patients, and patients with Gaucher disease. KC, MIP-1α, and MCP-1 were sequentially upregulated in plasma from FD mice. MCP-1, IL-10, IL-6, IL-12, and VEGF levels were elevated in plasma from Farber patients but not in control or JIA patients. C16-Ceramide (C16-Cer) and dhC16-Cer were upregulated in plasma from FD mice. a-OH-C18-Cer, dhC12-Cer, dhC24:1-Cer, and C22:1-Cer-1P accumulated in plasma from patients with FD. Most cytokines and only a-OH-C18-Cer returned to baseline levels in HSCT-treated Farber patients. Sphingosines were not altered. Chitotriosidase activity was also relatively low. A unique cytokine and ceramide profile was seen in the plasma of Farber patients that was not observed in plasma from HSCT-treated Farber patients, JIA patients, or Gaucher patients. The cytokine profile can potentially be used to prevent misdiagnosis of Farber as JIA and to monitor the response to treatment. Further understanding of why these signaling molecules and lipids are elevated can lead to better understanding of the etiology and pathophysiology of FD and inform development of future treatments.

Tissue biomarkers of drug efficacy: case studies using a MALDI-MSI workflow.

MALDI MS imaging (MALDI-MSI) offers a capability to not only evaluate the distribution, localization and metabolism of drugs within tissues but also allow correlative tissue measurement of the effect of the drug on biomolecules in the targeted pathway. Particularly for MALDI-MSI, lipid molecules are readily detectable within tissues. Case study examples are provided for two different drugs targeting the sphingosine-1-phosphate/ceramide nexus in tumor xenograft tissues. A workflow combining high-resolution MALDI-MSI with on-tissue confirmation of targeted compounds using a structural library and on-tissue enzymatic digestion strategy is described. Representative images of drug metabolite distribution that correlate to an increase or decrease in sphingosine-1-phosphate or ceramide species are provided.

Sphingosine 1-Phosphate Receptor 2 Regulates the Migration, Proliferation, and Differentiation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are a multipotent cell population acquired most prominently from bone marrow with the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, and others. MSCs demonstrate the capacity to home to sites of injury and contribute to tissue repair. Sphingosine 1-phosphate (S1P) is a biologically active sphingolipid impacting proliferation, apoptosis, inflammation, and angiogenesis with changes in S1P concentration providing significant implications for various disease conditions including cancer, diabetes, and cardiac disease. These functions are primarily mediated by interactions with 5 G-protein coupled S1P receptors (S1PR1-5). In this paper, we demonstrate that inhibition of S1PR2 results in increased MSC clonogenicity, migration, and proliferation; features dependent on Erk phosphorylation. Furthermore, decreased S1PR2 expression decreases the differentiation of MSCs into adipocytes and mature osteoblasts that may be the result of increased expression of MSC pluripotency factors including Nanog, Sox-9, and Oct-4. Inhibition of S1PR1 and S1PR3 in contrast does not impact MSC migration or Erk activation although increased proliferation is observed. In the study, we describe the essential role of S1PR2 in MSC differentiation pathways through modification of pluripotency factors. We propose a MAPK dependent mechanism through S1PR2 inhibition that promotes equally multipotent MSC proliferation.

Acid ceramidase promotes nuclear export of PTEN through sphingosine 1-phosphate mediated Akt signaling.

The tumor suppressor PTEN is now understood to regulate cellular processes at the cytoplasmic membrane, where it classically regulates PI3K signaling, as well as in the nucleus where multiple roles in controlling cell cycle and genome stability have been elucidated. Mechanisms that dictate nuclear import and, less extensively, nuclear export of PTEN have been described, however the relevance of these processes in disease states, particularly cancer, remain largely unknown. We investigated the impact of acid ceramidase on the nuclear-cytoplasmic trafficking of PTEN. Immunohistochemical analysis of a human prostate tissue microarray revealed that nuclear PTEN was lost in patients whose tumors had elevated acid ceramidase. We found that acid ceramidase promotes a reduction in nuclear PTEN that is dependent upon sphingosine 1-phosphate-mediated activation of Akt. We were further able to show that sphingosine 1-phosphate promotes formation of a complex between Crm1 and PTEN, and that leptomycin B prevents acid ceramidase and sphingosine 1-phosphate mediated loss of nuclear PTEN, suggesting an active exportin-mediated event. To investigate whether the tumor promoting aspects of acid ceramidase in prostate cancer depend upon its ability to export PTEN from the nucleus, we used enforced nuclear expression of PTEN to study docetaxel-induced apoptosis and cell killing, proliferation, and xenoengraftment. Interestingly, while acid ceramidase was able to protect cells expressing wild type PTEN from docetaxel, promote proliferation and xenoengraftment, acid ceramidase had no impact in cells expressing PTEN-NLS. These findings suggest that acid ceramidase, through sphingosine 1-phosphate, promotes nuclear export of PTEN as a means of promoting tumor formation, cell proliferation, and resistance to therapy.

Radiation-induced acid ceramidase confers prostate cancer resistance and tumor relapse.

Escape of prostate cancer (PCa) cells from ionizing radiation-induced (IR-induced) killing leads to disease progression and cancer relapse. The influence of sphingolipids, such as ceramide and its metabolite sphingosine 1-phosphate, on signal transduction pathways under cell stress is important to survival adaptation responses. In this study, we demonstrate that ceramide-deacylating enzyme acid ceramidase (AC) was preferentially upregulated in irradiated PCa cells. Radiation-induced AC gene transactivation by activator protein 1 (AP-1) binding on the proximal promoter was sensitive to inhibition of de novo ceramide biosynthesis, as demonstrated by promoter reporter and ChIP-qPCR analyses. Our data indicate that a protective feedback mechanism mitigates the apoptotic effect of IR-induced ceramide generation. We found that deregulation of c-Jun induced marked radiosensitization in vivo and in vitro, which was rescued by ectopic AC overexpression. AC overexpression in PCa clonogens that survived a fractionated 80-Gy IR course was associated with increased radioresistance and proliferation, suggesting a role for AC in radiotherapy failure and relapse. Immunohistochemical analysis of human PCa tissues revealed higher levels of AC after radiotherapy failure than those in therapy-naive PCa, prostatic intraepithelial neoplasia, or benign tissues. Addition of an AC inhibitor to an animal model of xenograft irradiation produced radiosensitization and prevention of relapse. These data indicate that AC is a potentially tractable target for adjuvant radiotherapy.

Interdiction of sphingolipid metabolism to improve standard cancer therapies.

Non-surgical therapies for human malignancies must negotiate complex cell signaling pathways to impede cancer cell growth, ideally promoting death of cancer cells while sparing healthy tissue. For most of the past half century, medical approaches for treating cancer have relied primarily on cytotoxic chemotherapeutics that interfere with DNA replication and cell division, susceptibilities of rapidly dividing cancer cells. As a consequence, these therapies exert considerable cell stress, promoting the generation of ceramide through de novo synthesis and recycling of complex glycosphingolipids and sphingomyelin into apoptotic ceramide. Radiotherapy of cancer exerts similar geno- and cytotoxic cell stresses, and generation of ceramide following ionizing radiation therapy is a well-described feature of radiation-induced cell death. Emerging evidence now describes sphingolipids as mediators of death in response to newer targeted therapies, cementing ceramide generation as a common mechanism of cell death in response to cancer therapy. Many studies have now shown that dysregulation of ceramide accumulation-whether by reduced generation or accelerated metabolism-is a common mechanism of resistance to standard cancer therapies. The aims of this chapter will be to discuss described mechanisms of cancer resistance to therapy related to dysregulation of sphingolipid metabolism and to explore clinical and preclinical approaches to interdict sphingolipid metabolism to improve outcomes of standard cancer therapies.

LCL124, a cationic analog of ceramide, selectively induces pancreatic cancer cell death by accumulating in mitochondria.

Treatment of pancreatic cancer that cannot be surgically resected currently relies on minimally beneficial cytotoxic chemotherapy with gemcitabine. As the fourth leading cause of cancer-related death in the United States with dismal survival statistics, pancreatic cancer demands new and more effective treatment approaches. Resistance to gemcitabine is nearly universal and appears to involve defects in the intrinsic/mitochondrial apoptotic pathway. The bioactive sphingolipid ceramide is a critical mediator of apoptosis initiated by a number of therapeutic modalities. It is noteworthy that insufficient ceramide accumulation has been linked to gemcitabine resistance in multiple cancer types, including pancreatic cancer. Taking advantage of the fact that cancer cells frequently have more negatively charged mitochondria, we investigated a means to circumvent resistance to gemcitabine by targeting delivery of a cationic ceramide (l-t-C6-CCPS [LCL124: ((2S,3S,4E)-2-N-[6'-(1″-pyridinium)-hexanoyl-sphingosine bromide)]) to cancer cell mitochondria. LCL124 was effective in initiating apoptosis by causing mitochondrial depolarization in pancreatic cancer cells but demonstrated significantly less activity against nonmalignant pancreatic ductal epithelial cells. Furthermore, we demonstrate that the mitochondrial membrane potentials of the cancer cells were more negative than nonmalignant cells and that dissipation of this potential abrogated cell killing by LCL124, establishing that the effectiveness of this compound is potential-dependent. LCL124 selectively accumulated in and inhibited the growth of xenografts in vivo, confirming the tumor selectivity and therapeutic potential of cationic ceramides in pancreatic cancer. It is noteworthy that gemcitabine-resistant pancreatic cancer cells became more sensitive to subsequent treatment with LCL124, suggesting that this compound may be a uniquely suited to overcome gemcitabine resistance in pancreatic cancer.

Differential expression of epithelial and mesenchymal proteins in a panel of prostate cancer cell lines.

PURPOSE: Epithelial to mesenchymal transition is an important process that results in increased cell migration, invasion and metastasis of many carcinomas. During epithelial to mesenchymal transition epithelial cells down-regulate cell-cell adhesion molecules (ie E-cadherin), up-regulate mesenchymal proteins (ie N-cadherin and cadherin-11), alter polarity, reorganize the cytoskeleton and become isolated. In combination this leads to greater motility. We investigated the role of E-cadherin and the associated catenin-protein complex in regulating epithelial to mesenchymal transition in prostate cancer progression. MATERIALS AND METHODS: The relative invasion index of prostate cancer cells was assessed by MTT based in vitro invasion assay. Immunoprecipitation and Western blot were done to determine cadherin-complex formation, and catenin and cadherin protein expression. RESULTS: Restoration of E-cadherin expression in nonE-cadherin expressing prostate cancer cells decreased invasive potential. However, in vitro invasive potential was tightly regulated by the interaction of cadherin proteins with the catenin complex. E and N-cadherin, cadherin-11, and the catenin proteins α, ß, γ and p120 are important for the downstream signaling associated with epithelial to mesenchymal transition in tumor cells. CONCLUSIONS: Restoration of epithelial specific proteins, such as E-cadherin, in tumor cells can inhibit invasion. However, invasion is a complex process regulated not only by E and N-cadherin but also by catenin-complex proteins. The complex signaling process associated with tumor invasion warrants further investigation since crosstalk between overlapping signaling pathways is involved in regulating prostate cancer invasion, metastasis and progression.

Efficient growth suppression and apoptosis in human laryngeal carcinoma cell line HEP-2 induced by an adeno-associated virus expressing human FAS ligand.

BACKGROUND: Apoptosis induced by Fas/FasL system has been proposed as a gene therapy methold for various cancers. METHODS: We used adeno-associated virus-expressing enhanced green fluorescent protein (EGFP)-human FasL (AAV-EGFP-hFasL) to deliver FasL into Hep-2 cells, cytotoxicity was detected by MTS assay , apoptosis was confirmed by flow cytometry. We also treated the xenograft of Hep-2 tumor in nude mice with intratumoral injection of AAV-EGFP-hFasL. The size of the xenograft, the apoptosis in the xenograft, and the survival rate of the inoculated mice were then evaluated. RESULTS: Hep-2 cells infected with AAV-EGFP-hFasL showed increased apoptosis rate and killing effect compared with AAV-EGFP-infected cells. In addition intratumoral injections of AAV-EGFP-hFasL into Hep-2 xenografts induced significant growth suppression of tumors. CONCLUSION: Our findings suggest that the introduction of FasL into head and neck squamous cell carcinoma may induce significant apoptosis, and adeno-associated virus may be a useful vehicle for gene therapy.

Genome characterization of the oleaginous fungus Mortierella alpina.

Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids.

Targeting sphingolipid metabolism in head and neck cancer: rational therapeutic potentials.

IMPORTANCE OF THE FIELD: Ceramide accumulation has been shown to be a conserved mechanism of apoptosis initiation in normal physiological processes as well as in response to cancer treatments. Therefore, it is unsurprising that many cancers develop aberrations of sphingolipid metabolism that prevent the accumulation of ceramide, whether by reduction of ceramide generation or by enhanced ceramide catabolism, particularly dangerous when catabolism leads to generation of pro-tumor sphingosine-1-phosphate and ceramide-1-phosphate. Numerous studies have now implicated dysregulation of sphingolipid metabolism in head and neck cancers. AREAS COVERED IN THIS REVIEW: This review highlights the importance of sphingolipid metabolism and brings sphingolipid metabolism to the forefront in the investigation of novel therapies for head and neck cancer. It reviews sphingolipid-centric therapies under investigation in preclinical and clinical trials of cancers of the head and neck. WHAT THE READER WILL GAIN: The roles of sphingolipids and sphingolipid metabolism in cancer are reviewed and the reader will be brought up to date with discoveries in the field of sphingolipid metabolism in head and neck cancer. TAKE HOME MESSAGE: As treatments for head and neck cancers are currently limited, the potential of targeting sphingolipid metabolism should be taken into consideration as we seek novel ways to combat this group of tumors.

Ceramide plays a prominent role in MDA-7/IL-24-induced cancer-specific apoptosis.

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) uniquely displays broad cancer-specific apoptosis-inducing activity through induction of endoplasmic reticulum (ER) stress. We hypothesize that ceramide, a promoter of apoptosis, might contribute to mda-7/IL-24 induction of apoptosis. Ad.mda-7-infected tumor cells, but not normal cells, showed increased ceramide accumulation. Infection with Ad.mda-7 induced a marked increase in various ceramides (C16, C24, C24:1) selectively in prostate cancer cells. Inhibiting the enzyme serine palmitoyltransferase (SPT) using the potent SPT inhibitor myriocin (ISP1), impaired mda-7/IL-24-induced apoptosis and ceramide production, suggesting that ceramide formation caused by Ad.mda-7 occurs through de novo synthesis of ceramide and that ceramide is required for mda-7/IL-24-induced cell death. Fumonisin B1 (FB1) elevated ceramide formation as well as apoptosis induced by Ad.mda-7, suggesting that ceramide formation may also occur through the salvage pathway. Additionally, Ad.mda-7 infection enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin in cancer cells. ASMase silencing by RNA interference inhibited the decreased cell viability and ceramide formation after Ad.mda-7 infection. Ad.mda-7 activated protein phosphatase 2A (PP2A) and promoted dephosphorylation of the anti-apoptotic molecule BCL-2, a downstream ceramide-mediated pathway of mda-7/IL-24 action. Pretreatment of cells with FB1 or ISP-1 abolished the induction of ER stress markers (BiP/GRP78, GADD153 and pospho-eIF2alpha) triggered by Ad.mda-7 infection indicating that ceramide mediates ER stress induction by Ad.mda-7. Additionally, recombinant MDA-7/IL-24 protein induced cancer-specific production of ceramide. These studies define ceramide as a key mediator of an ER stress pathway that may underlie mda-7/IL-24 induction of cancer-specific killing.

Role of CXC chemokine ligand 13 in oral squamous cell carcinoma associated osteolysis in athymic mice.

Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion; however, the molecular mechanisms of tumor osteolysis are unclear. In this study, we identified high level expression of chemokine ligand, CXCL13 and RANK ligand (RANKL) in OSCC cells (SCC1, SCC12 and SCC14a). OSCC cell-conditioned media (20%) induced osteoclast differentiation which was inhibited by OPG in peripheral blood monocyte cultures indicating that OSCC cells produce soluble RANKL. Recombinant hCXCL13 (10 ng/ml) significantly enhanced RANKL-stimulated osteoclast differentiation in these cultures. Trans-well migration assay identified that CXCL13 induces chemotaxis of peripheral blood monocytes in vitro which was inhibited by addition of anti-CXCR5 receptor antibody. Zymogram analysis of conditioned media from OSCC cells revealed matrix metalloproteinase-9 (MMP-9) activity. Interestingly, CXCL13 treatment to OSCC cells induced CXCR5 and MMP-9 expression suggesting an autocrine regulatory function in OSCC cells. To examine the OSCC tumor cell bone invasion/osteolysis, we established an in vivo model for OSCC by subcutaneous injection of OSCC cells onto the surface of calvaria in NCr-nu/nu athymic mice, which developed tumors in 4-5 weeks. muCT analysis revealed numerous osteolytic lesions in calvaria from OSCC tumor-bearing mice. Histochemical staining of calvarial sections from these mice revealed a significant increase in the numbers of TRAP-positive osteoclasts at the tumor-bone interface. Immunohistochemical analysis confirmed CXCL13 and MMP-9 expression in tumor cells. Thus, our data implicate a functional role for CXCL13 in bone invasion and may be a potential therapeutic target to prevent osteolysis associated with OSCC tumors in vivo.

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.