Lysophosphatidylcholine Acyltransferase1 Overexpression Promotes Oral Squamous Cell Carcinoma Progression via Enhanced Biosynthesis of Platelet-Activating Factor

<div><p>Background</p><p>The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs.</p><p>Methods</p><p>We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression.</p><p>Results</p><p>LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression.</p><p>Conclusion</p><p>LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.</p></div

Establishment of shLPCAT1-transfected cells.

<p>(<b>A</b>) Expression of <i>LPCAT1</i> mRNA in shMock- and shLPCAT1-transfected cells (SAS- and Ca9–22-derived transfectants). <i>LPCAT1</i> mRNA expression in shLPCAT1-transfected cells is significantly (*<i>p</i><0.05, Mann-Whitney <i>U</i> test) lower than in the shMock-transfected cells. (<b>B</b>) Immunoblot analysis of LPCAT1 protein in shMock- and shLPCAT1-transfected cells (SAS- and Ca9–22-derived transfectants). The LPCAT1 protein expression in shLPCAT1-transfected cells is decreased markedly compared with the shMock-transfected cells.</p

Effect of LPCAT1 knockdown on OSCC-derived cell lines.

<p>(<b>A, B</b>) Proliferation assay of shMock- and shLPCAT1-transfected cells (SAS- and Ca9–22-derived transfectants). To determine the effect of shLPCAT1 on cellular proliferation, shLPCAT1- and shMock-transfected cells were seeded in 6-cm dishes at a density of 1×10⁴ viable cells/well. Both transfected cells were counted on seven consecutive days. The cellular growth of shLPCAT1-transfected cells (SAS- and Ca9–22- derived transfectants) is inhibited significantly compared with the shMock-transfected cells after 5 days (120 hours). The results are expressed as the mean ±SEM of values from three assays. The asterisks indicate significant (*<i>p</i><0.05, Mann-Whitney <i>U</i> test) differences between the shLPCAT1- and shMock-transfected cells. (<b>C, D</b>) Migration assay of shMock- and shLPCAT1-transfected cells (SAS- and Ca9–22-derived transfectants). To evaluate the effect of LPCAT1 knockdown on migration, uniform wounds were made in confluent culture of the shLPCAT1- and shMock-transfected cells (SAS- and Ca9–22-derived transfectants) and the extent of closure was monitored visually every 3 hours for 24 hours. The mean value was calculated from data obtained from three separate chambers. The wound area was decreased significantly (*<i>p</i><0.05, Mann-Whitney <i>U</i> test) in the culture of shMock-transfected cells after 12 hours, whereas a gap remained in the shLPCAT1-transfected cells. (<b>E, F</b>) Invasiveness assay of shMock- and shLPCAT1-transfected cells (SAS- and Ca9–22-derived transfectants). To evaluate the effect of LPCAT1 knockdown on invasiveness, we seeded 2.5×10⁵ cells in the serum-free medium of a 0.8-μm polyethylene terephthalate membrane insert in a transwell apparatus and added serum-supplemented medium in the lower chamber as a chemoattractant. After incubation at 37°C for 48 hours, cells that penetrated through the pores were fixed, stained, and counted using a light microscope at ×100 magnification. The mean value was calculated from data obtained from three separate chambers. The number of shLPCAT1-transfected cells penetrating through the pores is decreased significantly (*<i>p</i><0.05, Mann-Whitney <i>U</i> test) compared with the shMock-transfected cells.</p

Correlation between clinicopathological parameters of patients with OSCC and LPCAT1 protein expression.

<p>* <i>p</i><0.05</p><p>Correlation between clinicopathological parameters of patients with OSCC and LPCAT1 protein expression.</p

Expression profiles of LPCAT1 in OSCC-derived cell lines and OSCC samples.

<p>(<b>A</b>) Quantification of <i>LPCAT1</i> mRNA levels in OSCC-derived cell lines by qRT-PCR analysis. To determine the mRNA expression status of <i>LPCAT1</i>, we performed qRT-PCR analysis using 9 OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, Sa3, SAS, Ca9–22, KOSC2, HO-1-N-1, and HO-1-u-1), and HNOKs. <i>LPCAT1</i> mRNA is significantly up-regulated in the nine OSCC-derived cell lines compared with the HNOKs. The data are expressed as the mean ±SEM of values from three assays (*<i>p</i><0.05, Mann-Whitney <i>U</i> test). (<b>B</b>) Immunoblot analysis of LPCAT1 in the OSCC-derived cells lines and HNOKs. To investigate the protein expression status of LPCAT1, we performed immunoblot analysis in the same OSCC-derived cell lines and HNOKs. The LPCAT1 protein expression level is significantly up-regulated in all OSCC-derived cell lines compared with the HNOKs. Densitometric LPCAT1 protein data are normalized to the GAPDH protein levels. The values are expressed as a percentage of the HNOKs. (<b>C</b>) IHC of LPCAT1 on primary OSCC samples. Representative IHC results are shown for LPCAT1 protein in normal oral tissue (a, b) and primary OSCCs (c, d). The original magnifications are 100×(a, c), and 400×(b, d). Strong LPCAT1 immunoreactivity is detected in the primary OSCCs. (<b>D</b>) The status of LPCAT1 protein expression in primary OSCCs (n = 55) and the normal counterparts. The LPCAT1 IHC scores are calculated as follows: IHC score = 1×(number of weakly stained cells in the field) + 2×(number of moderately stained cells in the field) + 3×(number of intensely stained cells in the fields). The LPCAT1 IHC scores for normal oral tissues range from 0.5 to 68.5 and that of primary OSCCs range from 23.7 to 205.9. The LPCAT1 protein expression levels in OSCCs are significantly (*<i>p</i><0.01, Mann-Whitney <i>U</i> test) higher than those in normal oral tissues.</p

Understanding the connection between platelet-activating factor, a UV-induced lipid mediator of inflammation, immune suppression and skin cancer

Lipid mediators of inflammation play important roles in several diseases including skin cancer, the most prevalent type of cancer found in the industrialized world. Ultraviolet (UV) radiation is a complete carcinogen and is the primary cause of skin cancer. UV radiation is also a potent immunosuppressive agent, and UV-induced immunosuppression is a well-known risk factor for skin cancer induction. An essential mediator in this process is the glyercophosphocholine 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine commonly referred to as platelet-activating factor (PAF). PAF is produced by keratinocytes in response to diverse stimuli and exerts its biological effects by binding to a single specific G-protein-coupled receptor (PAF-R) expressed on a variety of cells. This review will attempt to describe how this lipid mediator is involved in transmitting the immunosuppressive signal from the skin to the immune system, starting from its production by keratinocytes, to its role in activating mast cell migration in vivo, and to the mechanisms involved that ultimately lead to immune suppression. Recent findings related to its role in regulating DNA repair and activating epigenetic mechanisms, further pinpoint the importance of this bioactive lipid, which may serve as a critical molecular mediator that links the environment (UVB radiation) to the immune system and the epigenome