Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression

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Abstract

Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

Introduction

Over the past decade, significant interest has been shown in elucidating the roles of sphingolipids in cancer and in cancer therapy [1]. This is because levels of both the enzymes that regulate sphingolipid metabolism, as well as levels of sphingolipid metabolites, change significantly in various cancer tissues and in response to chemotherapeutic agents [2], [3], [4], [5]. Of all the sphingolipids, ceramide has received particularly wide attention due to its role in regulating a number of cellular events, including apoptosis and differentiation [6]. Ceramide levels have been shown to change in a number of tumor tissues, such as head and neck squamous cell carcinoma (HNSCC) [7], [8], [9].

Although ceramide is often referred to as if it is one lipid, it actually exists as a number of different species which differ in both their sphingoid base composition and in the acyl chain length of the fatty acid that is attached to the sphingoid base [10]. Recently, a gene family encoding for six distinct Ceramide Synthase (CerS) enzymes has been discovered [11] (previously known as longevity assurance gene homologs, Lass genes), with each member of the CerS family synthesizing ceramide with a relatively restricted acyl chain composition. Thus, CerS1 and CerS5 synthesize C18- and C16-ceramides, respectively [12], [13], [14] (where C18 and C16 refer to the acyl chain length of the ceramide), CerS2 synthesizes very long acyl chain ceramides (C22–24-ceramide) [15], CerS3 synthesizes C26-ceramides [16] and CerS4 synthesizes C18–22-ceramides [13].

In the current study, we examine mRNA expression levels, by real time quantitative polymerase chain reaction (qPCR), of the six CerS genes, in breast cancer tissues and compare levels with paired normal tissue from the same patients. We describe some significant differences in the cancer tissues, which showed elevated CerS2 and 6 mRNA expression. Moreover, an interesting correlation was obtained between CerS expression and levels of sphingosine kinase (SK) 1; sphingosine kinase is the enzyme that synthesizes the important first and second messenger, sphingosine 1-phosphate (S1P), which is believed to act in an opposite manner to ceramide such that ceramide is a pro-apoptotic lipid whereas S1P is a pro-survival lipid [17]. We discuss the relevance of these findings to breast cancer etiology and diagnosis.

Section snippets

Material and methods

RNA samples. RNA samples from 20 invasive ductal carcinoma patients were obtained from the Cancer Tissue Bank Research Centre, University of Liverpool, England. Patient data is shown in Table 1. RNA quality was evaluated using an Agilent Bioanalyser instrument, and only RNA with an integrity level above 7, or with a good running profile, was used.

cDNA synthesis. cDNA was synthesized using 200 ng RNA and the Reverse-iT first strand synthesis kit (Thermo Scientific), using random hexamers, with 30 

CerS expression in breast cancer tissue

Paired tissue samples were obtained from healthy and cancerous tissues from the same patients, and were chosen according to histological markers detected by microscopy (Table 1). Of the 20 RNA samples used, two were from patients in Bloom Richardson stage I, 13 were in stage II, and 5 were in stage III. The tumor size of nine of the patients was below or equal to 20 mm, whereas 11 patients had bigger tumors ranging from 25 to 140 mm. All of the patients were lymph node positive (ranging from 3 to

Discussion

The main finding of the current study is that CerS mRNA expression is altered in human breast cancer tissue compared to paired normal tissue from the same patients. Thus, breast cancer joins the growing list of cancers in which the sphingolipid/ceramide pathway is altered.

While this study was in preparation, a study was published showing changes in ceramide levels, measured by liquid chromatography tandem mass spectrometry (LC–MS/MS), in 43 malignant breast tumors, 21 benign breast tumors and

Acknowledgments

This work was supported by the Yeda fund of the Weizmann Institute, the Women’s Health Research Center of the Weizmann Institute and a U.S.–Israel Binational Science Foundation Grant. The authors would like to acknowledge the Cancer Tissue Bank Research Centre, University of Liverpool, UK from whom quality assessed tissue samples and associated clinical data used in this study were obtained. A.H. Futerman is the Joseph Meyerhoff Professor of Biochemistry in the Weizmann Institute of Science.

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