A B S T R A C T PurposePlasma levels of lysophospholipids were evaluated as potential biomarkers for colorectal cancer (CRC), where a highly reliable and minimally invasive blood test is lacking. Patients and MethodsPatients with CRC (n ϭ 133) and control subjects (n ϭ 125) were recruited through the Cleveland Clinic. Preoperative plasma samples were analyzed for lysophospholipid levels using liquid chromatography mass spectrometry in a blinded fashion. Participants were randomly divided in a 2:1 ratio into a "training set" (TS) and a "validation set" (VS). Logistic regression models were used in the TS to identify markers that best discriminated between CRC and controls. A cutoff point for the final discriminating model was developed using the receiver operating characteristic curve to achieve 95% specificity. All analyses were then independently validated in the VS. ResultsPlasma levels of several lysophosphatidylcholines (LPCs), including 18:1-and 18:2-LPC, were significantly decreased in CRC patients compared with controls (P Ͻ .001). A model based on total saturated LPC and the difference between the proportional amounts of 18:2-LPC and 18:1-LPC in the unsaturated LPC fraction was derived from the TS. This model achieved a sensitivity and specificity of 82% and 93%, respectively, in the VS. Overall, 118 (94%) of 125 control subjects and 113 (85%) of 133 CRC cases were correctly identified, including eight (89%) of nine CRC cases with stage T1 disease. ConclusionPercentage of 18:1-LPC or 18:2-LPC plasma levels compared with total saturated LPC levels, either individually or in combination, may represent potential biomarkers for CRC.

We have reported previously that levels of lysophosphatidic acid (LPA) are elevated in the blood and ascites from patients with ovarian cancer. LPA stimulates proliferation of ovarian cancer cells and has been proposed as an autocrine growth factor. Here, we show that a novel autocrine loop of LPA promotes the migration of ovarian cancer cells, which is a critical step of tumor metastasis. We report that laminin, but not other extracellular matrix proteins, induces LPA production in ovarian cancer cells. A neutralizing antibody against beta1 integrin and a calcium-independent phospholipase A2-specific inhibitor, HELSS, block both LPA production and the haptotactic activity of laminin. Exogenously added LPA restores the migratory ability of HEY ovarian cancer cells to laminin. These data suggest that laminin-induced cell migration is mediated by LPA. We further show that a specific receptor for LPA, LPA3, is required for mediating the chemotactic activity of LPA. In addition, we show that cytosolic PLA2 is required for cell migration and its activation is phosphatidylinositol-3 kinase-dependent. These findings have revealed a new mechanism of crosstalk between a beta1 integrin receptor and a G protein-coupled receptor.

Calcium-independent phospholipase A 2 (iPLA 2 ) plays a pivotal role in phospholipid remodeling and many other biological processes, including inflammation and cancer development. iPLA 2 can be activated by caspase-3 via a proteolytic process in apoptotic cells. In this study we identify novel signaling and functional loops of iPLA 2 activation leading to migration of non-apoptotic human ovarian cancer cells. The extracellular matrix protein, laminin-10/11, but not collagen I, induces integrin-and caspase-3-dependent cleavage and activation of overexpressed and endogenous iPLA 2 . The truncated iPLA 2 (amino acids 514 -806) generates lysophosphatidic acid and arachidonic acid. Arachidonic acid is important for enhancing cell migration toward laminin-10/11. Lysophosphatidic acid activates Akt that in turn acts in a feedback loop to block the cleavage of poly-(ADP-ribose) polymerase and DNA fragmentation factor as well as prevent apoptosis. By using pharmacological inhibitors, blocking antibodies, and genetic approaches (such as point mutations, dominant negative forms of genes, and siRNAs against specific targets), we show that ␤ 1 , but not ␤ 4 , integrin is involved in iPLA 2 activation and cell migration to laminin-10/ 11. The role of caspase-3 in iPLA 2 activation and cell migration are supported by several lines of evidence. 1) Point mutation of Asp 513 (a cleavage site of caspase-3 in iPLA 2 ) to Ala blocks laminin-10/11-induced cleavage and activation of overexpressed iPLA 2 , whereas mutation of Asp 733 to Ala has no such effect, 2) treatment of inhibitors or a small interfering RNA against caspase-3 results in decreased cell migration toward laminin-10/11, and 3) selective caspase-3 inhibitor blocks cleavage of endogenous iPLA 2 induced by laminin-10/11. Importantly, small interfering RNA-mediated down-regulation of endogenous iPLA 2 expression in ovarian carcinoma HEY cells results in decreased migration toward laminin, suggesting that our findings are pathophysiologically important.

We previously reported that lysophosphatidic acid (LPA) represents a potential biomarker for ovarian and other gynecologic cancers. To further improve the accuracy and potentially increase the sensitivity and specificity of the assay, we developed an electrospray ionization mass spectrometry (ESI‐MS)‐based method to analyze LPA and related lysophospholipids. LPA, lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS), and lysophosphatidylcholine (LPC) could be detected with high sensitivity (in low pmol range) using this method. Standard curves were established for quantitative analysis. LPA and closely related lysophospholipids isolated from thin‐layer chromatography (TLC) plates were analyzed directly by ESI‐MS. This ESI‐MS‐based assay allows simultaneous detection and quantitation of all different species of LPAs and LPIs in a sample over a range of at least 5–300 pmol. Moreover, this test was at least 50 times more sensitive when a multiple reaction monitoring (MRM) mode was used. Using these protocols in a limited set of analyses, we found that both LPA and LPI were elevated in patients with ovarian cancer.

Bioactive lysolipid levels might be sensitive markers for detecting gynecologic cancers, particularly ovarian cancer. The prognostic value of lysolipids in ascites is worth further investigation. Bioactive lysolipid molecules can affect both the proliferative and metastatic potentials of ovarian cancer cells; therefore, regulation of the production or degradation of these lipids and interception of the interaction between these lipids and their receptors could provide novel and useful preventative or therapeutic measures.