Cancer Research Meeting Calendar Telomeres
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Springett, G. M.
Right arrow Articles by Dupont, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Springett, G. M.
Right arrow Articles by Dupont, J.
[Cancer Research 65, 9415-9425, October 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Lysophosphatidic Acid Acyltransferase-ß Is a Prognostic Marker and Therapeutic Target in Gynecologic Malignancies

Gregory M. Springett1,2, Lynn Bonham6, Amanda Hummer3, Irina Linkov5, Dipika Misra2, Chia Ma2, Gabriella Pezzoni6, Stefano Di Giovine6, Jack Singer6, Hiroaki Kawasaki7, David Spriggs2, Robert Soslow4 and Jakob Dupont2

1 Drug Discovery Program, Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center, Tampa, Florida; Developmental Chemotherapy Laboratory, Departments of 2 Medicine, 3 Biostatistics, 4 Pathology and 5 Immunohistochemistry Core Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York; 6 Cell Therapeutics Incorporated, Seattle, Washington; and 7 Department of Neuropsychiatry, Graduate School of Medical Sciences Kyushu University, Fukuoka, Japan

Requests for reprints: Jakob Dupont, Developmental Chemotherapy Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-8388; Fax: 212-717-3214; E-mail: dupontj{at}mskcc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lysophosphatidic acid, the substrate for lysophosphatidic acid acyltransferase ß (LPAAT-ß), is a well-studied autocrine/paracrine signaling molecule that is secreted by ovarian cancer cells and is found at elevated levels in the blood and ascites fluid of women with ovarian cancer. LPAAT-ß converts lysophosphatidic acid to phosphatidic acid, which functions as a cofactor in Akt/mTOR and Ras/Raf/Erk pathways. We report that elevated expression of LPAAT-ß was associated with reduced survival in ovarian cancer and earlier progression of disease in ovarian and endometrial cancer. Inhibition of LPAAT-ß using small interfering RNA or selective inhibitors, CT32521 and CT32228, two small-molecule noncompetitive antagonists representing two different classes of chemical structures, induces apoptosis in human ovarian and endometrial cancer cell lines in vitro at pharmacologically tenable nanomolar concentrations. Inhibition of LPAAT-ß also enhanced the survival of mice bearing ovarian tumor xenografts. Cytotoxicity was modulated by diacylglycerol effectors including protein kinase C and CalDAG-GEF1. LPAAT-ß was localized to the endoplasmic reticulum and overexpression was associated with redistribution of protein kinase C-{alpha}. These findings identify LPAAT-ß as a potential prognostic and therapeutic target in ovarian and endometrial cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The diacyl glycerolipid, phosphatidic acid, is a versatile lipid second-messenger that functions as a cofactor in several critical signaling pathways that are operant in cancer cells. For example, the full activation of c-Raf-1 and B-raf is manifested only when phosphatidic acid physically interacts with a polybasic amino acid segment in these kinases (1). This interaction is required for the translocation of Raf kinases to the plasma membrane where they phosphorylate and activate their targets. Similarly, binding of phosphatidic acid to a polybasic domain in mTOR is essential for its activation (2). Hence, the role of phosphatidic acid is central to the regulation of proteins in both proliferative and survival pathways in tumor cells. Phosphatidic acid can be produced by the enzymatic conversion of phosphatidylcholine to phosphatidic acid and choline by phospholipase D (3). Alternatively, diacylglycerol can be phosphorylated by diacylglycerol kinase to produce phosphatidic acid. Finally, lysophosphatidic acid (LPA) can be acylated at the sn-2 position by lysophosphatidic acid acyltransferase (LPAAT) to generate phosphatidic acid.

Six human LPAAT isoforms have been identified and designated LPAAT-{alpha}, -ß, -{delta}, -{varepsilon}, -{gamma}, and -{zeta} (4, 5). LPAAT-{alpha} (AGPAT1) and LPAAT-ß (AGPAT2) have been studied in greatest detail thus far. LPAAT-{alpha} mRNA is ubiquitously expressed in humans, with highest levels in skeletal muscle (6). In contrast, the pattern of LPAAT-ß expression is more tissue-specific, with highest levels in the liver and heart (4). The 278–amino acid sequence of LPAAT-ß is 48% homologous to LPAAT-{alpha} (7). Inherited loss of function of LPAAT-ß causes congenital generalized lipodystrophy 1, which is also known as Berardinelli-Seip syndrome (8, 9). This autosomal recessive disorder is characterized by an almost complete lack of body fat and severe insulin resistance, and this phenotype indicates that the LPAAT isoforms are not physiologically redundant.

In contrast, increased expression of LPAAT-ß is associated with the malignant state. LPAAT-ß expression is differentially up-regulated in tumor cells compared with normal tissues in a wide variety of tumor cell types, including lung, breast, colon, prostate, and gliomas (10). Expression has also been identified in tumor stroma and in tumor-associated vascular endothelium. In comparison, the expression of LPAAT-{alpha} is uniform in normal and tumor tissues (4). Ectopic overexpression of LPAAT-ß stimulates the proliferation of LNCaP cells in low serum in vitro (10). Enhancement of proliferative capabilities has also been noted in human breast cancer cells (MCF-7) engineered to overexpress LPAAT-ß (11). Conversely, knockdown of LPAAT-ß expression in DU145 prostate cancer cells with small interfering RNA (siRNA) inhibits proliferation (10). With respect to its role in signaling, LPAAT-ß mRNA is able to cooperate with Ras and Raf to enhance Erk activation when injected into oocytes in the Xenopus meiotic maturation assay. Indeed, inhibition of LPAAT-ß expression with siRNA in mammalian cells suppresses basal Erk phosphorylation. Inhibition of LPAAT-ß with small-molecule antagonists prevents the translocation of Raf to the plasma membrane and subsequent Erk phosphorylation. These inhibitors also suppress the activation of proteins in the phosphoinositide-3-kinase (PI3K)/Akt pathway, including Akt, mTOR, and S6 kinase (12).

LPA, the substrate for LPAAT-ß, is a well-studied autocrine/paracrine signaling molecule which is secreted by ovarian cancer cells and is found at elevated levels in the ascites fluid of women with ovarian cancer (13, 14). LPA signaling may contribute to the accumulation of ascites by inducing the synthesis and release of vascular endothelial growth factor (15). LPA also regulates the proliferation, survival, adhesion, and migration of ovarian cancer cells in vitro, and its presence at high levels in serum may represent an early biomarker for ovarian cancer detection. These effects of LPA are a manifestation of its first-messenger role as an extracellular ligand for the Edg (endothelial differentiation gene) family of G protein–coupled receptors (16, 17). The signal transduction functions of intracellular pools of LPA and the phosphatidic acid that can be generated from it by LPAAT-ß are poorly understood. In view of the biological activities of LPA and phosphatidic acid in ovarian cancer, we sought to investigate the role of LPAAT-ß, an enzyme that can convert LPA to phosphatidic acid. In this report, we examine the expression of LPAAT-ß in tissue microarrays of ovarian and endometrial cancers of various histologies. We found that LPAAT-ß was expressed in 70% of serous ovarian cancers and that high expression was predictive of a poorer prognosis in ovarian and endometrial cancer. We then examined the effects of pharmacologic inhibition of LPAAT-ß by a family of isoform-selective small-molecule inhibitors. These molecules exhibited cytotoxic activity at nanomolar concentrations in vitro and were effective in prolonging survival in a murine model of peritoneal carcinomatosis with a human ovarian tumor xenograft. Finally, we examined the subcellular localization of LPAAT-ß in ovarian cancer cells and investigated the influence of LPAAT-ß expression on protein kinase C (PKC) signaling. We present a case for the development of LPAAT-ß as a new therapeutic target and prognostic marker in gynecologic cancers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and constructs. The following cell lines were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS): SK-OV-3, OVCAR3, SK-UT-1, SK-UT-1B, HeLa, and SK-LMS-1. MHOC59, AN3CA, and HEC-1-A cells were kindly provided by Jeff Boyd (Department of Medicine, Clinical Genetics Service and Department of Surgery, Gynecology Service, Memorial Sloan-Kettering Cancer Center, New York, NY) and propagated in RPMI 1640 with 10% FBS. IGROV1 was cultured as previously described (18). OVCA 420 and OVCA 433 were kindly provided by Robert Bast (Translational Research, M.D. Anderson Cancer Center, Houston, TX). SKOV3-CD1 was generated by infection of SK-OV-3 with a retroviral vector (MSCVneo-CalDAG-GEF1) engineered to express mouse CalDAG-GEF1 using methods essentially as described (19). Infected cells were selected in G418 500 µg/mL (Sigma, Inc., St. Louis, MO) for 2 weeks. MSCVneo-CalDAG-GEF1 was generated by inserting the full-length mouse CalDAG-GEF1 cDNA into the XhoI and EcoRI sites of retroviral vector MSCVneo (Clontech, Inc., Palo Alto, CA). The sequence of the complete insert was confirmed in both directions, and expression was confirmed by Western blotting with rabbit polyclonal anti-CalDAG-GEF antibodies (data not shown).

SKOV3-PKC{alpha} was generated by stable transfection of SK-OV-3 with 5 µg of pCMV-PKC{alpha}–enhanced green fluorescent protein (EGFP) DNA (Clontech), which contains a COOH-terminal fusion of PKC-{alpha} to EGFP. Transfections were done with the N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate (DOTAP) liposomal reagent (Roche, Inc., Nutley, NJ) according to the suppliers' instructions. Transfected cells were selected for 2 weeks in G418 (500 µg/mL). Fusions of the fluorescent protein DsRed to the COOH terminus and NH2 terminus of full-length human LPAAT-ß were generated by ligation to pDsRed-N1 and pDsRed-C1 (Clontech), respectively. A third fusion of EGFP to the COOH terminus of LPAAT-ß was also generated by ligation to pEGFP-N1 (Clontech). The resulting constructs (pDsRed-LPAATß, pLPAATß-DsRed, and pLPAATß-EGFP) were transiently transfected into SK-OV-3 or SKOV3-PKC{alpha} using the DOTAP method with 5 µg of DNA. SKOV3-AKT-Myr was generated by stable infection of SK-OV-3 with retroviral vector SR{alpha}-AKT-Myr (kindly provided by Alfonso Bellacosa, Fox Chase Cancer Center, Department of Microbiology and Immunology, Philadelphia, PA), which expresses a constitutively active myristoylated AKT mutant. SKOV3-vector control was generated by infection with empty SR{alpha} vector (20).

Western blot analysis. An LPAAT-ß–specific monoclonal antibody, MoAb 4B12 (Covance Research Products, Denver, PA), raised against amino acids 155 to 166 of human LPAAT-ß was used in immunoblotting experiments (10). Total protein extracts from cell lines in culture were prepared in lysis buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mmol/L MgCl2, protease inhibitor cocktail (Roche)] and 35 to 50 µg was run on 12% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% bovine serum albumin. Membranes were incubated with MoAb 4B12 at a dilution of 1:2,500 in PBS with 1% Tween 20 for 1 hour at room temperature. Blots were then probed with horseradish peroxidase–conjugated anti-mouse secondary antibody (Amersham, Inc., Piscataway, NJ) for 1 hour. Bound antibody was detected by enhanced chemiluminescence (Perkin-Elmer, Inc., Boston, MA). Western blots were sequentially probed with antibodies to actin (sc-1616) and mouse anti-goat IgG-horseradish peroxidase (sc-2354) from Santa Cruz, Inc., Santa Cruz, CA.

Tumor microarrays and immunohistochemistry. The generation of ovarian and endometrial tumor tissue microarrays is described in detail elsewhere (21, 22). Briefly, patients undergoing surgery for gynecologic malignancies at the Memorial Sloan-Kettering Cancer Center had their tumor specimens banked under an Institutional Review Board–approved tissue acquisition protocol after signing informed consent. For the ovarian tumor microarray, all cases of Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stage I or II epithelial ovarian cancer surgically staged at the Memorial Sloan-Kettering Cancer Center over a 20-year period (January 1980-December 2000) were identified. Patients with BRCA 1 mutations or a strong family history of breast or ovarian cancer were excluded. All remaining cases for which archival tissues were available were included. For the endometrial tumor array, all cases of patients undergoing surgery for endometrial cancer over a 10-year period (January 1991-December 2001) were selected. All samples were from tumor primary sites and were obtained before chemotherapy or radiation. Tumor microarrays were constructed using core needle biopsies of formalin-fixed paraffin-embedded tissue specimens from patients. Circular sections 0.6 mm in diameter were arrayed using a Beecher Instruments arrayer. The microarrays contained 129 endometrial cancer specimens and 87 ovarian tumor specimens. The clinical data were retrieved from a review of the medical records. For comparison, 14 normal ovaries were examined. These specimens were obtained from patients undergoing prophylactic oophorectomy (n = 3), resection of mucinous cystadenomas (n = 4), oophorectomy with benign epithelial inclusion cysts (n = 3), normal ovaries from patients with cervical cancer (n = 3), and normal ovary from a patient with endometrial cancer (n = 1). Specimens were processed using heat-induced epitope retrieval with citric acid (pH 6.0). Slides were stained with anti-LPAAT-ß monoclonal antibody 4B12 at a 1:10,000 dilution followed by biotinylated horse anti-mouse IgG (1:500) from VectorLabs Inc. (Burlingame, CA). Finally, horseradish peroxidase–conjugated streptavidin (1:500) was added, and the slides were developed with 3,3'-diaminobenzidine chromogen solution (DAKO, Inc., Carpinteria, CA). Tumor specimens were scored as LPAAT-ß-positive when displaying intense staining compared with adjacent normal tissue.

Confocal microscopy. Live SK-OV-3 and SKOV3-PKC{alpha} cells were plated in 0.17-mm-thick glass delta-T cover dishes (Bioptechs, Inc., Butler, PA) in complete medium and allowed to adhere for 24 hours. Cells were subsequently treated with phorbol 12-myristate 13-acetate (PMA; 200 nmol/L) or transfected with pDsRed-C1, pDsRed-LPAATß, pLPAATß-DsRed, or pLPAATß-EGFP. At indicated times posttreatment, the cells were imaged with a 63x water immersion objective on a Leica (DM IRE2) laser scanning dual photon inverted confocal microscope. Digitally captured images were assembled using Paint Shop Pro (Jasc Software, Inc., Ottawa, Ontario, Canada). As indicated, selected images were processed by deconvolution with AutoDeblur (AutoQuant Imaging, Inc., Troy, NY).

Compounds and drugs. The identification of LPAAT-ß inhibitors has been previously described (10, 12). Compounds were dissolved in DMSO at indicated concentrations. For in vitro experiments, cisplatin was obtained from Sigma and dissolved in PBS at a concentration of 1 mmol/L. Stock solutions were stored at –70°C for 2 weeks. For in vivo experiments, Platamine was obtained from Pharmacia and dissolved in saline at 1 mg/mL.

The c-Jun-NH2-kinase (JNK) selective inhibitor, SP600125 (Biomol, Inc., Plymouth Meeting, PA), was dissolved in DMSO and applied to cultures in complete medium at a final concentration of 20 µmol/L. PMA (Sigma) was added to complete medium (200 nmol/L in DMSO) as indicated.

Alamar blue viability assay. Cell viability was determined using the Alamar blue reagent (BioSource International, Inc., Camarillo, CA; ref. 18). Cells were plated in 96-well plates at 1 x 103 cells/well in 200 µL of complete medium with either vehicle (DMSO) or compounds at varying concentrations. Cells were incubated with compounds for 72 hours, and 25 µL of Alamar blue was then added. After an additional 6 hours of incubation, fluorescence intensity at 530 nm (excitation) and 590 nm (emission) was determined on a CytoFluor Series 4000 multi-well plate reader (PerSeptive Biosystems, Inc.). The percentage of growth inhibition was calculated as the ratio of the mean fluorescence of the treated cultures to control vehicle–treated cultures. Each point on the resulting viability curve is the mean of 16 replicates (n = 16). The Alamar blue assay is based on the reduction of the dye by the metabolic activity of living cells, which produces a colorimetric and fluorometric change in the dye.

Lysophosphatidic acid acyltransferase-ß small interfering RNA treatment of ovarian tumor cells. Several siRNAs targeting LPAAT-ß were synthesized by Dharmacon Research (Lafayette, CO). The sense strands for each siRNA duplex were as follows. LPAAT-ß-specific siRNA beginning at nucleotide 604, 5'-GUGGUGUACUCUUCCUUCUdTdT-3', LPAAT-ß 3'untranslated region beginning at nucleotide 953, 5'-UCCCGGCUUCCAAAUACCAdTdT-3', and EGFP control siRNA, 5'-GCAGCACGACUUCUUCAAGdTdT-3'. SK-OV-3 and IGROV1 cells were plated on 96-well plates and transfected with 25 nmol/L siRNAs in serum-free Opti-MEM I (Invitrogen, Carlsbad, CA) using OligofectAMINE transfection reagent according to the manufacturer's protocol (Invitrogen). Following 12 hours of incubation at 37°C, M5 medium with 30% FBS was added, and 3 days later, Alamar blue viability assay was done. As mock transfection, cells were exposed to OligofectAMINE alone in the absence of oligonucleotides.

For LPAAT-ß Western blot analysis, 1 x 106 SK-OV-3 and IGROV1 cells were plated on six-well plates and transfection was done with 200 nmol/L siRNA or the controls of OligofectAMINE or medium alone.

Animal procedures. Immunodeficient female CD1 nu–/nu– mice were obtained from Charles River Laboratories. IGROV-1 cells (2.5 x 106 cells/mouse) suspended in PBS were injected into the peritoneum of 6- to 8-week-old mice (n = 15) on day 0. LPAAT-ß inhibitor CT-32521 dissolved in 50 µL of DMSO was given i.p. at doses of 28 or 37 mg/kg/d on days 3 to 7. Alternatively, mice (n = 15) were treated with cisplatin in saline at a dose of 6 mg/kg/d on days 3, 7, and 11. Controls were treated with DMSO or saline vehicle. T/C% was calculated as: median survival of drug-treated groups / median survival of control vehicle–treated groups x 100. Mice were housed in microisolator cages with autoclaved feed. Animal procedures were done in accordance with institutional guidelines.

Statistical analysis. Survival as a function of time was estimated using Kaplan-Meier methodology. S-Plus Software (Insightful Corporation, Seattle, WA) was used for analysis of survival and the histologic marker, LPAAT-ß, and GraphPad Prism (GraphPad Software, Inc., San Diego, CA) software was used for the remaining analyses. Survival curves were compared using the log-rank test. Differences in LPAAT-ß expression and histology were assessed with Fisher's exact test. In murine experiments, compound- and vehicle-treated groups were compared using the log-rank test, and a P value of ≤0.05 was considered significant. Student's t test was used for RNAi experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lysophosphatidic acid acyltransferase-ß is up-regulated in gynecologic malignancies. In view of the ability of LPAAT-ß to promote cell proliferation and the transformed phenotype, we examined the expression of this protein in human gynecologic tumors and normal ovarian epithelium using an LPAAT-ß monoclonal antibody. We employed an ovarian tumor microarray comprised of 87 early-stage cancers and an endometrial cancer microarray of 129 tumors. The arrays were generated from the archival specimens of all patients who underwent surgery for ovarian and endometrial cancer over a 20- and 10-year period, respectively. These arrays contained carcinomas of various histologies as well as borderline neoplasms of low malignant potential. Nonmalignant ovarian epithelium was examined in 14 specimens from patients undergoing oophorectomy for prophylaxis, resection of benign cysts, or cervical cancer. In normal tissues, the ovarian epithelium was negative for LPAAT-ß expression (Fig. 1A). This was the case for the surface epithelium, tubal epithelium, and the epithelium of inclusion and paratubal cysts. Focally weak staining was seen in the ovarian stromal cells and in endothelial cells. In contrast, ovarian carcinomas displayed intense cytoplasmic staining primarily within the epithelial component (Fig. 1B and C). Expression was detected in the tumor stroma at lower levels. Overall, 38 of 87 (44%) ovarian tumors expressed LPAAT-ß (Table 1). The majority of aggressive papillary serous carcinomas (12 of 18; 67%), clear cell carcinomas (10 of 15; 67%), and endometrioid carcinomas (7 of 13; 54%) were LPAAT-ß-positive. In contrast, only 6 of 17 (35%) borderline and 1 of 17 (6%) mucinous neoplasms were positive. When all ovarian carcinomas were compared with borderline tumors, LPAAT-ß overexpression was significantly (P < 0.001) associated with the presence of carcinoma. The expression pattern of LPAAT-ß in endometrial tumors (Fig. 1D) and malignant mixed Mullerian tumors (Fig. 1E) was similar to that in ovarian tumors with intense staining of the epithelial component. Although only 15% (7 of 47) of endometrioid carcinomas of the endometrium were positive for LPAAT-ß, 50% of serous (P = 0.0036), 45% of clear cell (P = 0.0235), and 73% of malignant mixed Mullerian tumors (P = 0.0001) of the endometrium were positive (Table 1). In malignant mixed Mullerian tumors, the sarcomatous component also displayed strong staining. All human ovarian carcinoma cell lines tested had detectable LPAAT-ß expression by Western blot at varying levels (Fig. 1F, lanes 1-8). This was also true of MHOC-59 (lane 5), a primary culture of ovarian carcinoma from ascites fluid. There was no relationship between the sensitivity of the cell lines to platinum and the expression of LPAAT-ß (compare Fig. 1F, lanes 1 and 2 or 3 and 4). Endometrial and cervical carcinoma cell lines were also positive, whereas uterine leiomyosarcoma cell lines were very low or negative.



View larger version (120K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Expression of LPAAT-ß in human ovarian and endometrial tumors and cell lines. A-E, representative sections from a gynecologic tumor tissue microarray stained with anti-LPAAT-ß antibody are shown (B-E) in comparison with normal ovary (A). The epithelial component of each tumor is strongly positive. Stromal tissue is weakly positive in normal tissue and in tumors (magnification, x100). A, normal ovarian tissue. B, papillary serous ovarian carcinoma. C, clear cell carcinoma. D, endometrioid endometrial carcinoma. E, sarcomatous component of a malignant mixed mullerian tumor of the endometrium. F, expression of LPAAT-ß in human tumor cell lines by Western blot: (lanes 1-8) ovarian cancer cell lines; (1) A2780 CDDP-sensitive, (2) A2780 CDDP-resistant, (3) SKOV3 CDDP-sensitive, (4) SKOV3 CDDP-resistant, (5) MHOC59, (6) OVCAR3, (7) OVCA433, (8) OVCA420; (lanes 9 and 12) endometrial carcinoma cell lines; (9) AN3CA, (12) HEC1A; (lanes 10, 11, and 14) uterine sarcoma cell lines; (10) SKUT1, (11) SKLMS1, (14) SKUT1B, (13) cervical carcinoma cell line HeLa.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Expression of LPAAT-ß in ovarian and endometrial tumors

 
Expression of lysophosphatidic acid acyltransferase-ß correlates with prognosis. Because the expression of LPAAT-ß correlated with tumor histology, we asked whether the presence of this protein influenced clinical outcome. The medical records of patients with tumors included in the tissue microarrays were reviewed for disease status and survival data. Median follow-up for ovarian and endometrial cancer cases was 59 and 37 months, respectively. Analysis by Kaplan-Meier methods revealed that LPAAT-ß expression was associated with a significantly reduced progression-free survival (P = 0.009; Fig. 2A) and overall survival (death hazard ratio, 4.7; 95% confidence interval, 1.8-17.3; P = 0.01) in ovarian cancer (Fig. 2B). At 10 years, the overall survival of patients with LPAAT-ß-positive ovarian carcinoma was 58% compared with 93% for LPAAT-ß-negative tumors. It should be noted that the ovarian tumor microarray is comprised entirely of early-stage tumors (stage IA-IIC). Nevertheless, LPAAT-ß expression was able to differentiate between tumors with a 10-year overall survival of 90%, which is typical of early-stage disease and tumors with a substantially worse prognosis (58% overall survival) despite early stage. Similarly, progression-free survival was worse in LPAAT-ß-positive (P = 0.01) endometrial cancer (Fig. 2C). No statistically significant difference in overall survival was noted in endometrial cancer (Fig. 2D).



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Correlation of LPAAT-ß expression with clinical outcome. A and B, Kaplan-Meier univariate survival analysis of patients with early-stage (I, II) ovarian cancer. A, progression-free survival in ovarian cancer by LPAAT-ß status. B, overall survival in ovarian cancer by LPAAT-ß status. C and D, Kaplan-Meier univariate survival analysis of patients with endometrial cancer. C, progression-free survival in endometrial cancer by LPAAT-ß expression. D, overall survival in endometrial cancer by LPAAT-ß status. Insets, P values (log-rank test) comparing each pair of curves.

 
Small-molecule lysophosphatidic acid acyltransferase-ß inhibitors are cytotoxic to ovarian and endometrial carcinoma cell lines in vitro. The finding of differential up-regulation of LPAAT-ß expression in ovarian and endometrial tumors and the correlation of this expression with clinical outcome led us to test a translational research hypothesis—that selective inhibition of LPAAT-ß in gynecologic cancers would be an effective antitumor therapeutic strategy. To this end, we tested the activity of a family of isoform-selective small-molecule inhibitors of LPAAT-ß. These compounds were identified in a high-throughput chemical library screen for molecules that could inhibit LPAAT-ß in a cell-free biochemical assay (10). They are aryldiaminotriazines that display uncompetitive inhibition of sn-1-18:1 LPA binding to LPAAT-ß at nanomolar concentrations. They display no activity against LPAAT-{alpha} at concentrations of up to 40 µmol/L (12). The inhibitors do not inhibit phospholipase D1 hydrolysis, phospholipase D2 activity, or PI3K activity (12), thus, suggesting relative specificity for LPAAT-ß. We tested five of these first- (CT32521, CT32648) and second-generation (CT32228, CT32176, CT32615) LPAAT-ß inhibitors and one control compound (CT32212) with a similar structure but weak LPAAT-ß inhibitory activity in cell proliferation assays in vitro. Kill curves were generated using the fluorescence-based Alamar blue assay, which allows generation of kill curves without washing steps or manual counting of cells (23). Representative cytotoxicity curves are shown in Fig. 3A-D, where each point is the ratio of the mean fluorescence of a compound-treated culture to a vehicle (DMSO)–treated culture (n = 16 independent determinations within an experiment, two experiments). CT32228 is representative of this family of compounds with an IC50 (50% growth inhibitory concentration) of 50 and 104 nmol/L on human ovarian carcinoma cell lines SK-OV-3 and MHOC59, respectively (Fig. 3A and B). A similar range of activity was seen with human endometrial carcinoma cell lines AN3CA (IC50 = 37 nmol/L) and HEC-1A (IC50 = 100 nmol/L; Fig. 3C and D). A control compound without LPAAT-ß inhibitory activity, CT32212, was cytotoxic only at concentrations that were one to two orders of magnitude greater on these cell lines (IC50 ≥1,500-10,000 nmol/L). CT32228 exhibited little activity against the uterine leiomyosarcoma cell line, SK-LMS (Fig. 3E), which has little or no LPAAT-ß expression detectable by Western blot (Fig. 1F, lane 11). The relative potencies of the series of compounds used in this study were similar in all four cell lines tested (Fig. 3A-D). This allowed us to do a structure-function analysis to ascertain the critical determinants of antiproliferative activity against gynecologic cancer cell lines. The presence of substituents in the R3-NH-para-phenyl position (Fig. 3F) seems to be essential for activity because the positioning of groups in the R4 ortho-phenyl position as in CT32212 is associated with loss of activity. Aliphatic groups (CH2OH) at R3 were more potent than halogenated groups (Cl > Br > CF3). Similarly, aliphatic substitution at R1 (CH3CH2OH > CH3) was associated with greater activity. In our series, CT32615 with aliphatic substitutions at both R1 and R3 was the most active compound, with an IC50 as low as 25 nmol/L against MHOC59 ovarian carcinoma cells.



View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Structure and activity of LPAAT-ß inhibitors in ovarian and endometrial carcinoma cell lines in vitro. A-D, the antiproliferative effects of small-molecule LPAAT-ß inhibitors were tested against a panel of two ovarian, two endometrial, and one uterine leiomyosarcoma cell lines. A, SK-OV-3. B, MHOC59. C, AN3CA. D, HEC-1A. E, SK-LMS1. Compounds used were: CT32212 (green circle), CT32648 (blue diamond), CT32521 (red square), CT32228 (yellow triangle), CT32176 (lavender triangle), and CT32615 (solid pink circle). F, the compound structures are shown with corresponding IC50 values for each cell line indicated below. Curves and IC50 values are from a representative experiment (n = 2, with each point being the mean of 16 independent replicates).

 
Inhibition of lysophosphatidic acid acyltransferase-ß protein expression with small interfering RNA reduces ovarian cancer cell line viability. In order to confirm the biological effect of LPAAT-ß abrogation in SK-OV-3 and IGROV1 ovarian cancer cells, we used siRNAs directed against LPAAT-ß mRNA to inhibit protein expression. In these experiments, SK-OV-3 and IGROV1 cells transfected with LPAAT-ß siRNA1 (L1) had decreased expression of LPAAT-ß protein detectable by Western blot analysis (Fig. 4A). SK-OV-3 and IGROV1 cell lines transfected with the LPAAT-ß siRNAs (L1) also had diminished survival in the Alamar blue viability assay compared with cells transfected with control EGFP siRNA (P < 0.0001; Fig. 4B). No growth-inhibitory effect was observed in mock-transfected cells (O). This result was confirmed with a second LPAAT-ß siRNA2 (L2) directed against a distinct sequence. SK-OV-3 and IGROV1 cell survival was decreased by 48% and 50%, respectively, with these siRNAs (three experiments). Thus, both pharmacologic- and siRNA-mediated inhibition of LPAAT-ß activity leads to growth inhibition of ovarian tumor cells in vitro.



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. siRNA knockdown of LPAAT-ß in SK-OV-3 and IGROV1 cell lines. A, Western blot analysis of SK-OV-3 and IGROV1 cells transfected with LPAAT-ß siRNAs (L1 and L2), EGFP control siRNA (E), OligofectAMINE alone control (O), and medium alone control (U) using an LPAAT-ß-specific monoclonal antibody. Control Western blot analysis for ß-actin expression is also represented. B, SK-OV-3 and IGROV1 cells transfected with LPAAT-ß siRNAs (L1 and L2) inhibit cell viability in an Alamar blue viability assay compared with EGFP (E) control siRNA-transfected cells (P < 0.0001), mock-transfected (O), or untransfected (U) cells (n = 3 experiments).

 
Lysophosphatidic acid acyltransferase-ß inhibitors are effective against ovarian tumor xenografts in vivo. Peritoneal carcinomatosis is responsible for much of the morbidity and mortality associated with progressive ovarian cancer. Therefore, to test the efficacy of LPAAT-ß inhibition in vivo, we used a murine peritoneal carcinomatosis model (24). Immunodeficient nude mice were inoculated in the peritoneal cavity with 2.5 x 106 cells of the IGROV-1 human ovarian carcinoma xenograft. When injected i.p., these cells grow as rapidly progressive peritoneal carcinomatosis, which is typically fatal in 10 to 12 days. Inoculated mice (n = 8 per treatment group) were treated i.p. with either vehicle (DMSO) or LPAAT-ß inhibitor CT32521 at 37 mg/kg/d on days 3 to 7. Because the platinum drug class is the most clinically effective group of agents currently available against ovarian carcinoma, for comparison, we used a control group of mice (n = 10) treated i.p. on days 3, 7, and 11 with 6 mg/kg/d of cisplatin or saline vehicle. Survival by treatment group was analyzed by Kaplan-Meier methods and the results from a representative experiment (n = 3) are plotted in Fig. 5A and summarized in Fig. 5B. Mice treated with vehicle expired in 9 to 10 days, and mice treated with cisplatin experienced improved survival (median survival, 25 days; P < 0.0001). CT32521 treatment produced improved survival compared with vehicle (median survival, 19 days; P < 0.0001). CT32521 was not generally as effective as cisplatin in most experiments. Administering CT32521 at a lower dose (25 mg/kg/d) or on a 3-day schedule (days 3, 7, and 11) was not effective in comparison with vehicle (data not shown). CT32521 was well-tolerated, with only one toxic death out of 30 mice treated at the 37 mg/kg/d dose level. Two toxic deaths out of 35 mice treated with platinum were observed. We conclude that pharmacologic inhibition of LPAAT-ß activity is an effective therapeutic strategy in this preclinical animal model.



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Survival of an ovarian cancer xenograft mouse model treated with an LPAAT-ß inhibitor. A, athymic nude mice were inoculated with 2.5 x 106 IGROV-1 human ovarian carcinoma cells in the peritoneal cavity. Mice (n = 8) were treated with 37 mg/kg/d of CT32521 for 5 days (days 3-7) or DMSO (n = 8). As a positive control, another cohort of mice was also treated with 6 mg/kg/d of cisplatin (n = 10) or saline. Kaplan-Meier survival curves by treatment group are shown in (A). Survival curves of treated mice were compared with corresponding vehicle controls and P values calculated using the log rank test. B, summary of median survival and T/C (%) ratios (median survival of compound treated mice/median survival of vehicle treated mice x 100). T/C >120% is typically regarded as positive in National Cancer Institute protocol drug screens.

 
The cytotoxicity of lysophosphatidic acid acyltransferase-ß inhibition is modulated by diacylglycerol/phorbol ester signaling. Because phosphatidic acid may contribute to PI3K/AKT/mTOR signaling by facilitating activation of mTOR, we sought to determine whether LPAAT-ß inhibitors could influence survival signals transmitted by mutational activation of AKT. We found that overexpression of constitutive active myristoylated AKT (AKT-Myr) in SK-OV-3 ovarian carcinoma cells failed to produce any shift in the cytotoxicity curve and IC50 of CT32228 when compared with control SK-OV-3 cells (Fig. 6A). This is consistent with blockade of the pathway at a point distal to AKT by CT32228. In addition, we were unable to identify statistically significant differences in clinical outcome based on AKT or PTEN expression in our ovarian and endometrial tumor tissue arrays (data not shown). These findings led us to evaluate the role of other signaling proteins that might influence the sensitivity of tumor cells to LPAAT-ß inhibitors. Consistent with studies on myeloma cell lines, we found that treatment with the JNK-1, -2, -3–selective inhibitor SP600125 at 20 µmol/L slightly enhanced the resistance of SK-OV-3 cells to the cytotoxic effect of CT32228 (Fig. 6B; ref. 25). This effect did not achieve statistical significance, and there was no definitive correlation of the susceptibility to CT32228 and the levels of JNK-1 and -3 protein expression when examined by Western blotting in all cell lines used in Fig. 3 (data not shown).



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Diacylglycerol/phorbol ester signaling modulates the cytotoxicity of LPAAT-ß inhibitors. A, cytotoxicity of CT32228 to SK-OV-3 cells expressing a constitutively active myristoylated AKT mutant (AKT-Myr) or empty vector control. B, cytotoxicity to SK-OV-3 cells of CT32228 alone or CT32228 combined with JNK-selective inhibitor, SP600125 (20 µmol/L). C, cytotoxicity of CT32228 to control SK-OV-3 cells or SK-OV-3 overexpressing CalDAG-GEF1 protein (SKOV3-CD1) in the presence or absence of PMA (200 nmol/L). Comparing control SK-OV-3 to SKOV3-CD1 + PMA (P < 0.0001). D, cytotoxicity of CT32228 to control SK-OV-3 cells or SK-OV-3 overexpressing PKC-{alpha} protein (SKOV3-PKC{alpha}) in the presence or absence of PMA (200 nmol/L). Comparing control SK-OV-3 to SKOV3-PKC{alpha} + PMA (P < 0.0001). Representative kill curves are shown. Each point is the mean of 16 determinations (n = 3 experiments).

 
We were led to examine diacylglycerol effector pathways because phosphatidic acid and diacylglycerol are interconvertible by lipid phosphate phosphatase and diacylglycerol kinase (Fig. 7; ref. 26). Diacylglycerol effectors, such as PKC, have a well-described role in modulating the sensitivity of ovarian cancer cells to cytotoxic chemotherapy (27). In addition to PKC, nonkinase diacylglycerol effector proteins, such as the calcium- and diacylglycerol-regulated guanine nucleotide exchange factors (CalDAG-GEF), are able to regulate some aspects of malignant transformation (28–30). Accordingly, using transfection or retroviral transduction, we engineered derivatives of SK-OV-3, which constitutively overexpress either CalDAG-GEF1 (SKOV3-CD1; Fig. 6C) or PKC-{alpha} (SKOV3-PKC{alpha}; Fig. 6D). Both of these diacylglycerol effectors were able to enhance the resistance of SK-OV-3 cells to CT32228. This finding suggests that some portion of phosphatidic acid produced by LPAAT-ß might be converted to diacylglycerol and used for second-messenger signaling to PKC and CalDAG-GEF effectors. Indeed, treatment of control SK-OV-3 with phorbol ester (PMA), which is an exogenous diacylglycerol-mimetic, reduced the cytotoxicity of CT32228 and increased the IC50 from 50 to 60 to >100 nmol/L (Fig. 6C and D). This effect was more pronounced in SKOV3-CD1 (Fig. 6C) and SKOV3-PKC{alpha} (P < 0.0001; Fig. 6D). This implicates both kinase and nonkinase diacylglycerol effector systems in the proliferative/survival effects of LPAAT-ß signaling. The possibility that manipulating LPAAT-ß activity might influence PKC signaling is particularly intriguing because the expression of some PKC isoforms in ovarian tumors reportedly has prognostic implications (31). This might represent one mechanistic basis for the negative effect of LPAAT-ß expression on clinical outcome observed in Fig. 2.



View larger version (47K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 7. A model for the function of LPAAT-ß and phosphatidic acid in signaling. LPAAT-ß in endoplasmic reticulum membranes generates phosphatidic acid by addition of a second acyl chain to the sn-2 position of LPA. Phosphatidic acid within membranes binds and contributes to the full activation of mTOR and Raf. A portion of phosphatidic acid is converted to diacylglycerol by lipid phosphate phosphatase-3, which is also an integral membrane protein found in part in the endoplasmic reticulum. If polyunsaturated-diacylglycerol is generated, it may function as a second-messenger to PKC and/or CalDAG-GEF (RasGRP) family members. Conventional and novel PKC isoforms translocate to membranes in response to diacylglycerol stimulation, and CalDAG-GEF1 stimulates GTP/GDP exchange on Rap1 resulting in activation of Rap1. Rap1-GTP activates B-Raf and the MEK/Erk cascade. PKC can also regulate this pathway through its action on Ras/c-Raf.

 
Consequently, we next sought to obtain more direct evidence of an effect of LPAAT-ß expression on PKC function. Because the rapid translocation of conventional PKC isoforms in response to transient elevation of diacylglycerol or phorbol ester treatment can be observed in live cells using confocal microscopy with green fluorescent protein tags, we used EGFP-tagged PKC{alpha} as a diacylglycerol sensor (32). When treated with 200 nmol/L PMA for >5 minutes, SKOV3-PKC{alpha} cells displayed typical translocation of PKC{alpha}-EGFP primarily to the cell margins at the plasma membrane and to endoplasmic reticulum (Fig. 8A). With more prolonged exposure (>15 minutes), PKC{alpha} is known to be down-regulated in an ubiquitin-dependent process and the intensity of fluorescent signals accordingly decrease (data not shown; refs. 33, 34). SKOV3-PKC{alpha} cells transiently transfected with control constructs expressing DsRed protein over 12 to 24 hours acquired uniform cytoplasmic and nuclear red fluorescent signal (Fig. 8B, middle) with void areas consistent with absent expression in the endoplasmic reticulum. Within the cytoplasmic compartment, the distribution of PKC{alpha}-EGFP overlapped with DsRed (Fig. 8B, right) except in the endoplasmic reticulum where only the green signal of endoplasmic reticulum–localized PKC{alpha} was seen. Hence, transfection of DsRed alone did not affect the distribution of PKC{alpha}-EGFP under these conditions. When fusions of DsRed to either the COOH terminus (DsRed-LPAATß) or NH2 terminus (LPAATß-DsRed) of LPAAT-ß were expressed, punctate signals were observed throughout the cytoplasm in a pattern consistent with endoplasmic reticulum localization (Fig. 8C, middle). The patchy pattern is likely related to the propensity of the tetrameric DsRed protein to form aggregates when overexpressed as a fusion protein (35). Cells expressing DsRed-tagged LPAAT-ß displayed redistribution of PKC{alpha}-EGFP (Fig. 8C, left). Over 12 to 24 hours, PKC{alpha}-EGFP concentrated at the cell margins and at spots throughout the cytoplasm typical of the endoplasmic reticulum (Fig. 8C, left, top row). With COOH terminus-tagged LPAAT-ß at 48 to 72 hours, larger cytoplasmic aggregates were found in which PKC{alpha}-EGFP and LPAATß-DsRed colocalized (Fig. 8C, bottom row, right). Because of the tendency of DsRed fusions with LPAAT-ß to aggregate, we sought to confirm the subcellular localization of LPAAT-ß with a third fusion to EGFP, which, unlike DsRed, is monomeric. Using three-dimensional reconstruction of a stacked series of deconvoluted images of SK-OV-3 cells expressing LPAATß-EGFP revealed the reticular pattern of the endoplasmic reticulum (Fig. 8D). The most intense signal was in the perinuclear region consistent with the rough endoplasmic reticulum. A similar pattern has been noted in ECV-304 cells (10). Together, these findings are consistent with the notion that increased LPAAT-ß expression and activity can influence the subcellular distribution of PKC. A proposed model for this signaling circuitry is presented in Fig. 7.



View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 8. Localization of PKC-{alpha} in ovarian cancer cells expressing LPAAT-ß. A, SK-OV-3 cells constitutively expressing COOH-terminal EGFP-tagged PKC-{alpha} (SKOV3-PKC{alpha}) were treated with PMA 200 nmol/L for 0 minutes (left), 1 minute (middle), and 5 minutes (right). Arrows, sites of plasma membrane translocation (magnification, x63). B, SKOV3-PKC{alpha} cells were transfected with control vector expressing DsRed protein. PKC{alpha}-EGFP displays cytoplasmic and endoplasmic reticulum localization (left). DsRed is also distributed in the cytoplasm (middle) and nucleus. The cytoplasmic distribution of PKC{alpha}-EGFP is not altered and overlaps with that of DsRed (right). C, SKOV3-PKC{alpha} cells transfected with NH2-terminal DsRed-tagged LPAAT-ß (SKOV3-DsRed-LPAATß) display redistribution of PKC{alpha} to the cell margins (arrows) and endoplasmic reticulum 12 hours posttransfection (left, top row). DsRed-LPAATß is found in scattered aggregates (middle). Cells in the same field without strong red signals maintain a uniform cytoplasmic PKC{alpha} pattern (right, top row). SKOV3-PKC{alpha} cells transfected with COOH-terminal DsRed-tagged LPAAT-ß (SKOV3-LPAATß-DsRed) display large aggregates in a pattern consistent with endoplasmic reticulum distribution (left and middle, bottom row) in which PKC{alpha} and LPAAT-ß colocalize (right, bottom row; magnification, x100). D, SK-OV-3 cells expressing COOH-terminal EGFP-tagged LPAAT-ß were imaged as a stacked series, deconvoluted and three-dimensional reconstructed. LPAAT-ß is distributed throughout the rough endoplasmic reticulum in the perinuclear region and the smooth endoplasmic reticulum peripherally.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
With the completion of the Human Genome Project, the task of selecting which of 30,000+ proteins are appropriate therapeutic targets and/or prognostic markers for clinical translation is formidable. Although there is no unanimity of opinion on what the criteria for target validation should be, certain requirements are clearly desirable (36). The candidate target must be produced in the specified tumor type. Ideally, the target should be differentially expressed in tumors compared with normal tissue. The function of the target should contribute to the initiation and/or maintenance of some aspect of the malignant phenotype. Finally, inhibition of the target's function in a pharmacologically tenable manner should be cytotoxic or at least cytostatic to cancer cells. We have presented evidence that LPAAT-ß is overexpressed in ovarian and endometrial carcinomas relative to normal tissue. Those patients with LPAAT-ß-positive tumors had a poor clinical outcome compared with patients whose tumors were negative for LPAAT-ß expression. The ovarian tumors in our tissue microarray were from patients with early-stage disease. In an examination of LPAAT-ß expression in a panel of stage III to IV patients, we have found that 68% are positive.8 Further study will be needed to determine whether the negative prognostic implications of LPAAT-ß expression extend to patients with advanced-staged disease, which is the most common presentation. Nevertheless, the ability of LPAAT-ß expression to differentiate between early-stage ovarian cancer patients who will do well and those who will be at higher risk for poor outcome would be clinically useful as those patients may benefit from adjuvant therapy despite having early-stage tumors. The discriminative capability of LPAAT-ß expression in this population and the correlation with high-grade (papillary serous) histology also implies that this protein may influence critical biological features of ovarian cancer cells, such as adhesion, migration, invasion, and angiogenesis, which might contribute to early dissemination of disease. We further showed that inhibition of LPAAT-ß enzymatic activity with small-molecule inhibitors, exemplified by CT32228, is cytotoxic to ovarian and endometrial cancer cells at nanomolar concentrations in vitro and can prolong the survival of mice inoculated with human ovarian tumor xenografts. That siRNA knockdown of LPAAT-ß also inhibits the viability of ovarian cancer cells supports the conclusion that it is the inhibition of this target that is the mechanism by which CT32228 kills cancer cells. The relative resistance to CT32228 of SK-LMS, which has little detectable LPAAT-ß protein expression, would suggest that this leiomyosarcoma cell line is not dependent on LPAAT-ß activity for continued proliferation and survival. However, we have noted that the relative expression of LPAAT-ß protein does not correlate closely with the relative sensitivity of the cell lines to these inhibitors (compare Fig. 1F, lanes 3 and 5 or 9 and 12 to Fig. 3A and B or C and D, respectively). Similarly, enforced overexpression of LPAAT-ß in ovarian carcinoma cell line with a retroviral vector did not produce a significant change in sensitivity to several of these inhibitors.9 This suggests that it may be the extent of dependence of a particular tumor on LPAAT-ß that controls the sensitivity to the inhibitors rather than the absolute expression level alone. This finding is not unprecedented, and in the era of targeted cancer therapeutics, it is a recurring theme. For example, the expression level of estrogen receptor correlates poorly with tamoxifen sensitivity in breast cancer cells that also express HER2/neu (37). Similarly, sensitivity to anti-Her2 monoclonal antibody trastuzumab in breast cancer cells does not correlate with expression (38). Lack of correlation between tumor target expression and drug sensitivity also exists for the proteosome inhibitor PS-341 (bortezomib; ref. 39), the topoisomerase I inhibitor CPT-11 (irinotecan; ref. 40), and the epidermal growth factor receptor–targeting drugs C225 (erbitux) or ZD1839 (Iressa; ref. 41). Only recently has the beginning of a molecular understanding of epidermal growth factor receptor–targeting drugs been reported (42).

With regard to LPAAT-ß, we have examined both expression and enzymatic activity of the tumor cells and its relationship to the IC50 of the inhibitory compounds. As with all the instances above, the relationship is not simple (e.g., cells with twice the expression/activity are twice as resistant). Possible mechanistic reasons for the lack of correlation could be signaling interference, counter-regulatory mechanisms, relative differences in the dependence of a given cell line on the pathway or enhanced cellular drug efflux. Based on these findings, we conclude that LPAAT-ß is a valid target for clinical translation in gynecologic malignancies.

It has become increasingly clear that lipids like LPA and phosphatidic acid are much more than intermediates in membrane biosynthesis. Phosphatidic acid also functions as a cofactor in the activation of multiple signaling molecules including Raf, PKC-{zeta}, phosphoinositol 4-kinase, phospholipase C-{gamma}, and mTOR (Fig. 7; refs. 1, 2, 43–45). Considering this dual role, the question arises as to whether there are distinct subcellular pools of phosphatidic acid that are used for different purposes and to what extent these pools intermingle. If phosphatidic acid pools used for signal transduction and membrane biosynthesis are in equilibrium, this potentially presents an opportunity to manipulate the quantity of phosphatidic acid available for signaling by targeting the metabolic enzymes that can directly or indirectly contribute to phosphatidic acid homeostasis and thereby influence cell growth and survival. This approach has been shown in principle with respect to other metabolites in the biosynthetic pathway, such as LPA and diacylglycerol. For example, overexpression of plasma membrane localized lipid phosphate phosphatase can dephosphorylate extracellular LPA and thereby attenuate its positive effect on ovarian cancer cell proliferation, migration, and invasiveness (46, 47). Similar observations have been made with respect to diacylglycerol where activation of PKC and CalDAG-GEF family members can be dampened by overexpression of diacylglycerol kinase, which converts available diacylglycerol to phosphatidic acid (48, 49). Alternatively, overexpression of diacylglycerol acyl transferase reduces total cellular diacylglycerol by converting it to triglyceride, which inhibits proliferation and anchorage-independent growth in transformed fibroblasts (50). Our findings suggest that by depriving ovarian cancer cells of a source of phosphatidic acid from LPAAT-ß, the survival of these cells can be undermined. Until recently, it was not appreciated that the phosphatidic acid supplied by LPAAT-ß could contribute to signaling. This role had been ascribed largely to phosphatidylcholine-phospholipase D, which can generate phosphatidic acid from phosphatidylcholine. The fatty-acyl chains in acyl-phosphatidic acid generated by phospholipase D are composed largely of saturated and monounsaturated lipids (51). Whereas these saturated-phosphatidic acid species seem to function in signaling, the saturated-diacylglycerol derived from interconversion do not activate PKC (52, 53). The fatty-acyl chain composition of acyl-phosphatidic acid generated by LPAAT-ß seems to be similar to that generated by phospholipase D.10 If this is the case, then diacylglycerol produced indirectly from LPAAT-ß would be mostly saturated and destined for use in membrane biosynthesis, and the effects of LPAAT-ß on the distribution of PKC{alpha} observed here would be through an indirect mechanism. For example, phosphatidic acid from LPAAT-ß may contribute to the activation of phospholipase C-{gamma}, which then produces polyunsaturated-diacylglycerol for signaling. Further biochemical studies will test these models. A LPAATß-phosphatidic acid-PKC signaling axis would potentially be clinically relevant to a number of malignancies where PKC expression has prognostic importance. For example, PKC-{iota} expression is positively correlated with ovarian tumor grade and median survival (31). In human ovarian carcinoma cell lines, changes in PKC-{alpha} expression are associated with sensitivity to chemotherapy (27). PKC-{alpha} expression is inversely correlated with estrogen receptor status in breast and endometrial cancers and may be associated with tamoxifen treatment failure in breast cancer (54, 55). Differential expression of PKC-{alpha} and -ß has been noted in early prostate carcinoma compared with adjacent benign prostatic epithelium (56). It remains to be clarified which of the isoforms of PKC identified in ovarian and endometrial carcinomas are most relevant to the pathogenesis of these malignancies. In the studies reported here, PKC-{alpha} was selected as a representative member of the conventional class. Early phase I and II trials of PKC-{alpha} inhibitors in ovarian cancer have been disappointing and suggest that other isoforms may be more appropriate targets in this disease (57, 58). Determination of the influence of LPAAT-ß signaling on these isoforms would be important in devising potential combination targeted therapies against both LPAAT-ß and PKC.


   Acknowledgments
 
Grant support: G.M. Springett is an Amos Fellow of the Robert Wood Johnson Foundation and is supported by grants from the American Society of Clinical Oncology and NIH T32CA09207. D.R. Spriggs is supported by NIH grants K24CA087933, P01CA052477, and R01CA084009. J. Dupont is supported in part by grants from the American Society of Clinical Oncology (Career Development Award), the Cancer Research Institute, and the Damon Runyon-Lilly Clinical Investigator Award Program.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the Memorial Sloan-Kettering Cancer Center molecular cytology core for assistance with the confocal microscopy studies, Mario Leitao for assistance in generating tissue microarrays, and Jeni Moses for assistance in compound screening. We are grateful to David Hollenback for critically reading the manuscript and Michael Wakelam for helpful discussion.


   Footnotes
 
8 Unpublished data. Back

9 L. Bonham, unpublished data. Back

10 David Hollenback, personal communication. Back

Received 2/15/05. Revised 7/26/05. Accepted 8/ 5/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Andresen BT, Rizzo MA, Shome K, Romero G. The role of phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade. FEBS Lett 2002;531:65–8.[CrossRef][Medline]
  2. Fang Y, Vilella-Bach M, Bachmann R, Flanigan A, Chen J. Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science 2001;294:1942–5.[Abstract/Free Full Text]
  3. Cummings R, Parinandi N, Wang L, Usatyuk P, Natarajan V. Phospholipase D/phosphatidic acid signal transduction: role and physiological significance in lung. Mol Cell Biochem 2002;234–5:99–109.
  4. Leung DW. The structure and functions of human lysophosphatidic acid acyltransferases. Front Biosci 2001;6:D944–53.[Medline]
  5. Li D, Yu L, Wu H, et al. Cloning and identification of the human LPAAT-{zeta} gene, a novel member of the lysophosphatidic acid acyltransferase family. J Hum Genet 2003;48:438–42.[CrossRef][Medline]
  6. Agarwal AK, Garg A. Congenital generalized lipodystrophy: significance of triglyceride biosynthetic pathways. Trends Endocrinol Metab 2003;14:214–21.[CrossRef][Medline]
  7. Eberhardt C, Gray PW, Tjoelker LW. cDNA cloning, expression and chromosomal localization of two human lysophosphatidic acid acyltransferases. Adv Exp Med Biol 1999;469:351–6.[Medline]
  8. Agarwal AK, Arioglu E, De Almeida S, et al. AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome 9q34. Nat Genet 2002;31:21–3.[CrossRef][Medline]
  9. Magre J, Delepine M, Khallouf E. Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13. Nat Genet 2001;28:365–70.[CrossRef][Medline]
  10. Bonham L, Leung DW, White T, et al. Lysophosphatidic acid acyltransferase-ß: a novel target for induction of tumour cell apoptosis. Expert Opin Ther Targets 2003;7:643–61.[Medline]
  11. Bonham L, Hollenback D, Leung D, White T, Morrison D, Finney RE. Validation of lysophosphatidic acid acyltransferase-b (LPAAT-b) as a novel target for anti-cancer drug discovery and development. Proc Am Assoc Cancer Res 2001;42:3608.
  12. Coon M, Ball A, Pound J, et al. Inhibition of lysophosphatidic acid acyltransferase ß disrupts proliferative and survival signals in normal cells and induces apoptosis of tumor cells. Mol Cancer Ther 2003;2:1067–78.[Abstract/Free Full Text]
  13. Fang X, Schummer M, Mao M, et al. Lysophosphatidic acid is a bioactive mediator in ovarian cancer. Biochim Biophys Acta 2002;1582:257–64.[Medline]
  14. Mills GB, Moolenaar WH. The emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer 2003;3:582–91.[CrossRef][Medline]
  15. Hu YL, Tee MK, Goetzl EJ, et al. Lysophosphatidic acid induction of vascular endothelial growth factor expression in human ovarian cancer cells. J Natl Cancer Inst 2001;93:762–8.[Abstract/Free Full Text]
  16. Goetzl EJ, Graeler M, Huang MC, Shankar G. Lysophospholipid growth factors and their G protein-coupled receptors in immunity, coronary artery disease, and cancer. Scientific World Journal 2002;2:324–38.
  17. Huang MC, Lee HY, Yeh CC, et al. Induction of protein growth factor systems in the ovaries of transgenic mice overexpressing human type 2 lysophosphatidic acid G protein-coupled receptor (LPA2). Oncogene 2004;23:122–9.[CrossRef][Medline]
  18. Benard J, Da Silva J, De Blois MC, et al. Characterization of a human ovarian adenocarcinoma line, IGROV1, in tissue culture and in nude mice. Cancer Res 1985;45:4970–9.[Abstract/Free Full Text]
  19. Springett GM, Moen RC, Anderson S, Blaese RM, Anderson WF. Infection efficiency of T lymphocytes with amphotropic retroviral vectors is cell cycle dependent. J Virol 1989;63:3865–9.[Abstract/Free Full Text]
  20. Bellacosa A, Chan TO, Ahmed NN, et al. Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene 1998;17:313–25.[CrossRef][Medline]
  21. Leitao MM, Soslow RA, Baergen RN, Olvera N, Arroyo C, Boyd J. Mutation and expression of the TP53 gene in early stage epithelial ovarian carcinoma. Gynecol Oncol 2004;93:301–6.[CrossRef][Medline]
  22. Dupont J, Wang X, Marshall DS, et al. Wilms tumor gene (WT1) and p53 expression in endometrial carcinomas: a study of 130 cases using tissue microarrays. Gynecol Oncol 2004;94:449–55.[CrossRef][Medline]
  23. Ahmed SA, Gogal RM, Jr., Walsh JE. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J Immunol Methods 1994;170:211–24.[CrossRef][Medline]
  24. Pratesi G, Tortoreto M, Corti C, Giardini R, Zunino F. Successful local regional therapy with topotecan of intraperitoneally growing human ovarian carcinoma xenografts. Br J Cancer 1995;71:525–8.[Medline]
  25. Hideshima T, Chauhan D, Hayashi T, et al. Antitumor activity of lysophosphatidic acid acyltransferase-ß inhibitors, a novel class of agents, in multiple myeloma. Cancer Res 2003;63:8428–36.[Abstract/Free Full Text]
  26. Brindley DN, English D, Pilquil C, Buri K, Ling ZC. Lipid phosphate phosphatases regulate signal transduction through glycerolipids and sphingolipids. Biochim Biophys Acta 2002;1582:33–44.[Medline]
  27. Isonishi S, Ohkawa K, Tanaka T, Howell SB. Depletion of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances platinum drug sensitivity in human ovarian carcinoma cells. Br J Cancer 2000;82:34–8.[CrossRef][Medline]
  28. Ebinu JO, Bottorff DA, Chan EY, et al. RasGRP, a Ras guanyl nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Science 1998;280:1082–6.[Abstract/Free Full Text]
  29. Kawasaki H, Springett GM, Toki S, et al. A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia. Proc Natl Acad Sci U S A 1998;95:13278–83.[Abstract/Free Full Text]
  30. Springett GM, Kawasaki H, Spriggs DR. Non-kinase second-messenger signaling: new pathways with new promise. Bioessays 2004;26:730–8.[CrossRef][Medline]
  31. Weichert W, Gekeler V, Denkert C, Dietel M, Hauptmann S. Protein kinase C isoform expression in ovarian carcinoma correlates with indicators of poor prognosis. Int J Oncol 2003;23:633–9.[Medline]
  32. Sakai N, Sasaki K, Ikegaki N, Shirai Y, Ono Y, Saito N. Direct visualization of the translocation of the {gamma}-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein. J Cell Biol 1997;139:1465–76.[Abstract/Free Full Text]
  33. Leontieva OV, Black JD. Identification of two distinct pathways of protein kinase C{alpha} down-regulation in intestinal epithelial cells. J Biol Chem 2004;279:5788–801.[Abstract/Free Full Text]
  34. Lu Z, Liu D, Hornia A, Devonish W, Pagano M, Foster DA. Activation of protein kinase C triggers its ubiquitination and degradation. Mol Cell Biol 1998;18:839–45.[Abstract/Free Full Text]
  35. Soling A, Simm A, Rainov N. Intracellular localization of Herpes simplex virus type 1 thymidine kinase fused to different fluorescent proteins depends on choice of fluorescent tag. FEBS Lett 2002;527:153–8.[CrossRef][Medline]
  36. Lindsay MA. Target discovery. Nat Rev Drug Discov 2003;2:831–8.[CrossRef][Medline]
  37. Shou J, Massarweh S, Osborne CK, et al. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. J Natl Cancer Inst 2004;96:926–35.[Abstract/Free Full Text]
  38. Nahta R, Takahashi T, Ueno NT, Hung MC, Esteva FJ. P27(kip1) down-regulation is associated with trastuzumab resistance in breast cancer cells. Cancer Res 2004;64:3981–6.[Abstract/Free Full Text]
  39. Chauhan D, Li G, Shringarpure R, et al. Blockade of Hsp27 overcomes Bortezomib/proteasome inhibitor PS-341 resistance in lymphoma cells. Cancer Res 2003;63:6174–7.[Abstract/Free Full Text]
  40. Candeil L, Gourdier I, Peyron D, et al. ABCG2 overexpression in colon cancer cells resistant to SN38 and in irinotecan-treated metastases. Int J Cancer 2004;109:848–54.[CrossRef][Medline]
  41. Janmaat ML, Kruyt FA, Rodriguez JA, Giaccone G. Response to epidermal growth factor receptor inhibitors in non-small cell lung cancer cells: limited antiproliferative effects and absence of apoptosis associated with persistent activity of extracellular signal-regulated kinase or Akt kinase pathways. Clin Cancer Res 2003;9:2316–26.[Abstract/Free Full Text]
  42. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39.[Abstract/Free Full Text]
  43. Ghosh S, Moore S, Bell RM, Dush M. Functional analysis of a phosphatidic acid binding domain in human Raf-1 kinase: mutations in the phosphatidate binding domain lead to tail and trunk abnormalities in developing zebrafish embryos. J Biol Chem 2003;278:45690–6.[Abstract/Free Full Text]
  44. Hekman M, Hamm H, Villar AV, et al. Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts. J Biol Chem 2002;277:24090–102.[Abstract/Free Full Text]
  45. Lim HK, Choi YA, Park W, et al. Phosphatidic acid regulates systemic inflammatory responses by modulating the Akt-mammalian target of rapamycin-p70 S6 kinase 1 pathway. J Biol Chem 2003;278:45117–27.[Abstract/Free Full Text]
  46. Tanyi JL, Hasegawa Y, Lapushin R, et al. Role of decreased levels of lipid phosphate phosphatase-1 in accumulation of lysophosphatidic acid in ovarian cancer. Clin Cancer Res 2003;9:3534–45.[Abstract/Free Full Text]
  47. Tanyi JL, Hasegawa Y, Lapushin R, et al. The human lipid phosphate phosphatase-3 decreases the growth, survival, and tumorigenesis of ovarian cancer cells: validation of the lysophosphatidic acid signaling cascade as a target for therapy in ovarian cancer. Cancer Res 2003;63:1073–82.[Abstract/Free Full Text]
  48. Pettitt TR, Wakelam MJ. Diacylglycerol kinase epsilon, but not {zeta}, selectively removes polyunsaturated diacylglycerol, inducing altered protein kinase C distribution in vivo. J Biol Chem 1999;274:36181–6.[Abstract/Free Full Text]
  49. Topham MK, Prescott SM. Diacylglycerol kinase {zeta} regulates Ras activation by a novel mechanism. J Cell Biol 2001;152:1135–43.[Abstract/Free Full Text]
  50. Bagnato C, Igal RA. Overexpression of diacylglycerol acyltransferase-1 reduces phospholipid synthesis, proliferation, and invasiveness in simian virus 40-transformed human lung fibroblasts. J Biol Chem 2003;278:52203–11.[Abstract/Free Full Text]
  51. Hodgkin MN, Pettitt TR, Martin A, Michell RH, Pemberton AJ, Wakelam MJO. Diacylglycerols and phosphatidates: which molecular species are intracellular messengers? Trends Biochem Sci 1998;23:200–4.[CrossRef][Medline]
  52. Lin P, Fung WJ, Gilfillan AM. Phosphatidylcholine-specific phospholipase D-derived 1,2-diacylglycerol does not initiate protein kinase C activation in the RBL 2H3 mast-cell line. Biochem J 1992;287:325–31.
  53. Pettitt TR, Marin A, Horton T, Liossis C, Lord JM, Wakelam MJ. Diacylglycerol and phosphatidate generated by phospholipases C and D, respectively, have distinct fatty acid compositions and functions. Phospholipase D-derived diacylglycerol does not activate protein kinase C in porcine aortic endothelial cells. J Biol Chem 1997;272:17354–9.[Abstract/Free Full Text]
  54. Fournier DB, Chisamore M, Lurain JR, Rademaker AW, Jordan VC, Tonetti DA. Protein kinase C {alpha} expression is inversely related to ER status in endometrial carcinoma: possible role in AP-1-mediated proliferation of ER-negative endometrial cancer. Gynecol Oncol 2001;81:366–72.[CrossRef][Medline]
  55. Tonetti DA, Morrow M, Kidwai N, Gupta A, Badve S. Elevated protein kinase C{alpha} expression may be predictive of tamoxifen treatment failure. Br J Cancer 2003;88:1400–2.[CrossRef][Medline]
  56. Cornford P, Evans J, Dodson A, et al. Protein kinase C isoenzyme patterns characteristically modulated in early prostate cancer. Am J Pathol 1999;154:137–44.[Abstract/Free Full Text]
  57. Advani R, Peethambaram P, Lum BL, et al. A Phase II trial of aprinocarsen, an antisense oligonucleotide inhibitor of protein kinase C {alpha}, administered as a 21-day infusion to patients with advanced ovarian carcinoma. Cancer 2004;100:321–6.[Medline]
  58. Yuen AR, Halsey J, Fisher GA, et al. Phase I study of an antisense oligonucleotide to protein kinase C-{alpha} (ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 1999;5:3357–63.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
K. Takeuchi and K. Reue
Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis
Am J Physiol Endocrinol Metab, June 1, 2009; 296(6): E1195 - E1209.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
X. Ye
Lysophospholipid signaling in the function and pathology of the reproductive system
Hum. Reprod. Update, September 1, 2008; 14(5): 519 - 536.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
P. Sabbatini and D. R. Spriggs
Consolidation for Ovarian Cancer in Remission
J. Clin. Oncol., February 1, 2006; 24(4): 537 - 539.
[Full Text] [PDF]

Home page
 Cancer Res.Home page
Targeting LPAAT-{beta} in Gynecologic Cancer
Cancer Res., December 15, 2005; 65(24): 11785 - 11785.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Springett, G. M.
Right arrow Articles by Dupont, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Springett, G. M.
Right arrow Articles by Dupont, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online