Clinical relevance of ceramide metabolism in the pathogenesis of human head and neck squamous cell carcinoma (HNSCC): Attenuation of C18-ceramide in HNSCC tumors correlates with lymphovascular invasion and nodal metastasis

doi:10.1016/j.canlet.2007.06.003

Cancer Letters

Volume 256, Issue 1, 18 October 2007, Pages 101-111

https://doi.org/10.1016/j.canlet.2007.06.003 Get rights and content

Abstract

It has been documented previously that defects in the generation of C₁₈-ceramide, a product of ceramide synthase 1 (CerS1), also known as longevity assurance gene 1 (hLASS1), play important roles in the pathogenesis and/or progression of HNSCC. However, whether altered levels of ceramide generation in HNSCC tumors have any clinical relevance remains unknown. In this study, the levels of endogenous ceramides were measured in tumor tissues of 45 HNSCC patients as compared to their normal tissues using high-pressure liquid chromatography/mass spectrometry (LC/MS), and then possible link between ceramide levels and the clinical parameters of HNSCC were examined. The data showed that the levels of C₁₆-, C₂₄-, C_24:1-ceramides were significantly elevated in the majority of tumor tissues compared to their normal tissues, while the levels of only C₁₈-ceramide were significantly decreased in HNSCC tumors, especially in tumor tissues of male patients. Importantly, it was also shown here that decreased C₁₈-ceramide levels in HNSCC tumor tissues were significantly associated with the higher incidences of lymphovascular invasion, and pathologic nodal metastasis. Importantly, attenuation of C₁₈-ceramide was also positively linked to the higher overall stages of the primary HNSCC tumors. Therefore, these data suggest, for the first time, that the defects in the generation/accumulation of C₁₈-ceramide might have important clinical roles in HNSCC, especially in lymphovascular invasion and nodal disease.

Introduction

Squamous cell carcinomas of the head and neck (HNSCC) are among the most aggressive group of cancers. Despite emerging new surgical techniques and chemoradiation protocols, HNSCC remains among the five leading causes of solid tumor-related deaths in the United States [1]. Five-year survival rates of patients with advanced stages of HNSCC remain around 50%, with little improvement for several decades. Although several tissue biomarkers such as p16, p53, cyclin D1, cyclo-oxygenase 2 (COX-2), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and matrix metalloproteinases have been associated with HNSCC, there is still a need for new diagnostic and/or prognostic markers [2].

The biologically active sphingolipid ceramide has been a source of interest in the regulation of cancer cell growth and response to therapy. In addition to its important role as a key molecule in sphingolipid metabolism, ceramide also has important effector functions, which involve the regulation of apoptosis, cell cycle arrest, or senescence [3].

Previous studies have demonstrated a role for sphingolipids in the regulation of HNSCC growth and progression. For example, ceramide and sphingosine have been shown to inhibit EGF receptor kinase in epidermoid cell A431 carcinoma cells [4], [5]. In addition, altering the levels of membrane glycolipids of human A431 cells by an inhibitor of glucosylceramide synthase resulted in a rapid loss of epithelial cell morphology, a reduced rate of cell growth, and inhibition of cell–substrate interaction [6]. In another study, treatment of squamous cell carcinoma cell line, DJM-1, with an exogenous ceramide promoted differentiation, and inhibited proliferation, suggesting a regulatory role of ceramide in growth and differentiation of keratinocytes [7]. Moreover, there have been additional studies which confirmed the active regulatory role of ceramide in HNSCC cells regarding the induction of apoptosis [8], EGF receptor modulation [9], [10], reversing drug and radiation resistance [11], [12], inhibition of neo-vascularization [13], and enhancing the anti-cancer actions of various chemotherapy agents [14], [15], or photodynamic therapy [16].

Recently, treatment of human UM-SCC-22A (SCC of the hypopharynx) cells with a novel exogenous ceramide analogue, l-threo-C₆-pyridinium-ceramide (L-t-C₆-Pyr-Cer) resulted in a significant inhibition of telomerase activity, and prevented the growth of HNSCC xenografts in SCID mice in vivo[17]. It is well recognized that telomerase is active in 80–90% of HNSCC, which is an essential marker for cancer cell immortalization. Telomerase is also associated with poor prognosis of patients with HNSCC [18], [19].

In another line of investigation, the functions of specific subspecies of endogenous ceramides with different fatty acid chain length in HNSCC growth and/or progression has been examined. Analysis of the levels of endogenous ceramides between tumor and normal mucosa tissues of the same patients with HNSCC showed that only C₁₈-ceramide levels were decreased in the majority of tumor tissues, whereas the levels of other ceramides, such as C₁₆- and C₂₄-ceramides were increased in HNSCC tumor tissues when compared to their normal counterparts [20]. Further experiments showed that overexpression of the mammalian homologue of the yeast longevity assurance gene 1 (LASS1), also referred to as ceramide synthase1 (CerS1), which is known to selectively generate C₁₈-ceramide, resulted in the inhibition of HNSCC cell growth, and enhanced chemotherapy-induced apoptosis in UMSCC22A cells in situ, and in HNSCC xenografts in vivo[20], [21].

Although, these data demonstrated an important role for ceramide signaling in the regulation of HNSCC pathogenesis and/or progression both in situ and in vivo, the clinical relevance of endogenous ceramide levels in HNSCC is still unknown. Therefore, in this study, we examined the association between changes in the levels of endogenous ceramide and clinical parameters of HNSCC. The data demonstrated that alterations of the C₁₈-ceramide levels in HNSCC tumors are significantly correlated with the presence of lymphovascular invasion and nodal metastatic disease, suggesting that attenuation of C₁₈-ceramide is highly associated with the advanced HNSCC in the clinic.

Section snippets

Clinical samples

With the permission of the Institutional Review Board, randomized tissue samples of 33 male (73%) and 12 female (27%) HNSCC patients were obtained from the tumor bank of the Hollings Cancer Center at Medical University of South Carolina. For each patient, paired tissue samples, obtained from the tumor or from the pathologically negative healthy mucosa near tumor site, were studied. Therefore, randomization was performed among the patients whose paired tissue specimens included both the

The levels of endogenous ceramides in HNSCC tumor tissues as compared to normal tissues

To examine the clinical relevance of ceramide, first the levels of endogenous ceramides were measured in 45 pairs of tissues (normal as compared to tumor tissues) obtained from patients with HNSCC using LC/MS, and results are presented in Table 2. Consistent with the previously published data [20], which were obtained from a smaller cohort (n = 14 pairs), the current data in this study using a larger cohort of patients (n = 45 pairs) showed that endogenous C₁₆-, and to a lesser extent C₂₄-, C_24:1

Discussion

In this report, the clinical relevance of endogenous ceramides in 45 pairs of HNSCC tumor and non-cancerous (normal) tissues was examined. Consistent with the previous data [20], results presented here demonstrated that the levels of only C₁₈-ceramide was significantly lower in the tumor tissues of patients with HNSCC, whereas the levels of other major ceramides, especially C₁₆-, and C₂₄-ceramides, were higher in these tumors, when compared to their normal counterparts. Interestingly, levels of

Acknowledgments

We thank the members of Ogretmen, Hannun, and Norris laboratories for their helpful discussions. This study was supported by research grants from the National Institutes of Health DE01657 (B.O.), and CA097132 (Y.A.H.), Department of Defense, phase VII program project grant through Hollings Cancer Center (B.O.), and the National Science Foundation/EPSCoR, EPS-0132573 (B.O.). The animal facility used in this study was supported by the National Institutes of Health, Grant Number C06 RR015455

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Clinical relevance of ceramide metabolism in the pathogenesis of human head and neck squamous cell carcinoma (HNSCC): Attenuation of C18-ceramide in HNSCC tumors correlates with lymphovascular invasion and nodal metastasis

Abstract

Introduction

Section snippets

Clinical samples

The levels of endogenous ceramides in HNSCC tumor tissues as compared to normal tissues

Discussion

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

Exp. Cell Res.

J. Biol. Chem.

Cancer Lett.

J. Photochem. Photobiol. B.

Otolaryngol. Head Neck Surg.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Clin. Epidemiol.

J. Voice

Oral Oncol.

Am. J. Otolaryngol.

Int. J. Radiat. Oncol. Biol. Phys.

Am. J. Surg.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

Cancer statistics

CA Cancer J. Clin.

Molecular predictors of clinical outcome in patients with head and neck squamous cell carcinoma

Int. J. Exp. Pathol.

Biologically active sphingolipids in cancer pathogenesis and treatment

Nat. Rev. Cancer

Glycolipid depletion using a ceramide analogue (PDMP) alters growth, adhesion, and membrane lipid organization in human A431 cells

J. Cell Physiol.

Keratinocyte differentiation is induced by cell-permeant ceramides and its proliferation is promoted by sphingosine

Arch. Dermatol. Res.

Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis

Biochem. J.

Clinical relevance of ceramide metabolism in the pathogenesis of human head and neck squamous cell carcinoma (HNSCC): Attenuation of C₁₈-ceramide in HNSCC tumors correlates with lymphovascular invasion and nodal metastasis