[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer is the main cause of death from gynaecological malignancies. In spite of the efficacy of platinum-paclitaxel treatment in patients with primary epithelial ovarian carcinoma, platinum-based chemotherapy is not curative and resistance remains one of the most important causes of treatment failure. Although ABC transporters have been implicated in cellular resistance to multiple drugs, the clinical relevance of these efflux pumps is still poorly understood. Thus, we examined the prognostic role of transporters of the MRP family (i.e., ABCC1/MRP1, ABCC4/MRP4) to gain insights into their clinical impacts. A case material of 127 patients with ovarian carcinoma at different stages and histotypes was used. The expression of MRP1 and MRP4 was examined by immunohistochemistry using tissue microarrays in tumor specimens collected at the time of initial surgery expression. We found an association between MRP1 expression and grading, and we observed that MRP4 displayed an unfavourable impact on disease relapse in multivariate analysis (HR = 2.05, 95% CI: 1.01–4.11;
). These results suggest that in epithelial ovarian cancer, MRP1 may be a marker for aggressiveness because its expression was associated with tumor grade and support that MRP4 may play an unfavourable role in disease outcome.
[Show abstract][Hide abstract] ABSTRACT: A major challenge in advanced-stage epithelial ovarian cancer (EOC) is prediction of chemoresistant relapse. Our aim was to identify a microRNA (miRNA) signature associated with early relapse in advanced-stage EOC patients. miRNA expression was assessed by microarray profiling in training (n = 55) and test (n = 30) sets selected on the basis of time to relapse (TTR), followed by internal quantitative reverse transcriptase-PCR validation on a set of 45 consecutive cases unselected for clinical response and external in silico validation on publicly available datasets. Thirty-two differentially expressed miRNAs in early vs. late relapsing patients were identified in the training set. In the test set, 8 of these, belonging to a cluster located on chrXq27.3, were down-modulated in early relapsing patients. Hierarchical clustering of the internal validation set according to chrXq27.3 miRNA expression associated low miRNA expression with shorter TTR (log-rank P=0.00074, HR 2.44). The cluster was an independent prognostic factor in both internal and external validation sets. Forced expression of chrXq27.3-cluster selected miRNAs in human EOC cellular models was associated to reduction of cell proliferation and increased sensitivity to cisplatin. The role of down-modulation of the chrXq27.3 miRNA cluster in early relapse of advanced-stage EOC patients and its association to a reduced sensitivity to chemotherapeutic treatments warrant further investigation.
[Show abstract][Hide abstract] ABSTRACT: The impairment of apoptotic pathways represents an efficient mechanism to promote chemoresistance in cancer cells. We previously showed that in epithelial ovarian cancer (EOC) cells, long isoform of cellular FLICE-inhibitory protein (c-FLIP(L)) accounts for apoptosis resistance in a context of functional p53 and resistance could be overcome by c-FLIP(L) downmodulation. Here, we studied the association between c-FLIP(L) and p53 expressions and their prognostic impact in EOC patients. Tumor tissue from 207 patients diagnosed with primary EOC was analyzed by immunohistochemistry (IHC) for c-FLIP(L) and p53 expressions, and multiple correspondence analysis (MCA) was used to evaluate the multivariable pattern of association among patients' clinical-pathological characteristics and biological determinants. IHC revealed c-FLIP(L) expression and p53 nuclear accumulation inversely related (P = 0.0001; odds ratio = 0.29, confidence interval (CI) = 0.15-0.055). MCA indicated that p53 accumulation was associated to clinical-pathological variables, while c-FLIP(L) expression contributed to the overall association pattern independently from other's clinical characteristics and complementary to p53. Kaplan-Meier curves showed a reduced survival time according to c-FLIP(L) expression in concert with p53 accumulation (median overall survival (OS): 35 months) compared with lack of expression of both markers (median OS: 110 months; log-rank test, P value = 0.024). The multivariable Cox regression model, adjusted for known prognostic factors, identified c-FLIP(L) expression, but not p53 nuclear accumulation, as an independent prognostic factor for adverse outcome (hazard ratio = 1.82, 95% CI = 1.17-2.82; P = 0.008). Altogether these data support the independent contribution of c-FLIP(L) in refining the prognostic information obtained from standard clinical-pathological indicators, confirming its pivotal role in promoting cell survival.
Endocrine Related Cancer 04/2009; 16(2):443-53. DOI:10.1677/ERC-08-0218 · 4.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Elucidation of the mechanisms responsible for aberrant phosphatidylcholine (PC) metabolism in cancer cells may allow identification of novel biomarkers of tumor progression and design of new targeted anticancer therapies. We recently reported up-regulation of PC-specific phospholipases in epithelial ovarian cancer cells (EOC) compared with nontumoral (normal or immortalized) counterparts (EONT). In the present study, we focused, in the same cell systems, on levels, subcellular localization, and activity of PC-specific phospholipase C (PC-PLC), for which a key role in cell proliferation, differentiation, and apoptosis has been shown in several mammalian cells. A 66-kDa PC-PLC isoform, detected in nuclear and cytoplasmic compartments of both EOC and EONT cells, accumulated on the external plasma membrane of cancer cells only, where it colocalized with beta1 integrin, in nonraft membrane domains. PC-PLC activity was 3-fold higher in total cell lysates and 5-fold higher in membrane-enriched fractions of EOC compared with EONT cells. Serum deprivation induced in EOC, but not in EONT, cells a 3-fold decrease in PC-PLC activity, associated with a 40% drop in S-phase fraction. The recovery of both variables to their original levels in serum-restimulated (or lysophosphatidic acid-restimulated) EOC cells was strongly delayed, for at least 24 h, in the presence of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609). The S-phase of serum-restimulated EONT cells was not sensitive to D609. These findings warrant further investigations on the role of PC-PLC and on the effects of its inhibition on the pathways responsible for constitutive EOC cell stimulation and cell proliferation.
Cancer Research 09/2008; 68(16):6541-9. DOI:10.1158/0008-5472.CAN-07-6763 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Currently available clinicopathologic prognostic factors are imperfect predictors of clinical course in advanced-stage epithelial ovarian cancer patients. New molecular predictors are needed to identify patients with higher risk of relapse or death from disease. In a retrospective study, we investigated the prognostic impact of activated leukocyte cell adhesion molecule (ALCAM) expression in epithelial ovarian cancer.
We analyzed the effect of cell-anchorage loss on ALCAM cellular localization in vitro and assessed ALCAM expression by immunohistochemistry in a series of 109 well-characterized epithelial ovarian cancer patient samples. Chi-square test, Kaplan-Meier method, and Cox proportional hazard analyses were used to relate ALCAM cellular localization to clinical-pathologic parameters and to overall survival (OS) rate.
Loss of epithelial ovarian cancer cell anchorage was associated both in vitro and in vivo with decreased ALCAM membrane expression. In vivo, ALCAM was localized to cell membrane in normal surface ovarian epithelium, whereas in 67% of the epithelial ovarian cancer samples, membrane localization was decreased or even lost, and the molecule was mainly expressed in cytoplasm. Median OS in this group of patients was 58 months, whereas a median OS was not yet reached in patients with ALCAM membrane localization (P = 0.036, hazard ratio [HR] = 2.0, 95% confidence interval [CI] 1.1 to 3.5). In a multivariate Cox regression model including all the available clinicopathologic variables, loss of ALCAM membrane expression was an independent factor of unfavorable prognosis (P = 0.042, HR = 2.15, 95% CI: 1.0 to 4.5).
Decreased/lost ALCAM membrane expression is a marker of poorer outcome in epithelial ovarian cancer patients and might help to identify patients who could benefit from more frequent follow-up or alternative therapeutic modalities.
Clinical Cancer Research 04/2008; 14(6):1726-33. DOI:10.1158/1078-0432.CCR-07-0428 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Novel Ab-based immunotherapeutic strategies have exploited T-cell receptor-like chimeric immune receptors (CIR) expressed on the surface of transduced human peripheral blood mononuclear cell (PBMC) to redirect potent non-major histocompatibility complex-dependent cytotoxicity to tumor cells expressing a tumor-associated antigens. We transduced human PBMC with 2 fully human CIRs that trigger through the zeta-chain of CD3 and contain either one of two human scFv specific for the same epitope on the extracellular domain of HER2 but with distinctly different affinities (KD 1616 and 1 nM) for this antigen. Potent direct CIR-mediated killing and in vitro tumor growth inhibition mediated by transduced PBMC were observed against targets expressing different levels of HER2. High-affinity CIR showed stronger ability to bind Ag and retain binding than low-affinity CIR. When lytic potential of the 2 CIRs was evaluated, their efficiency was comparable under conditions of high CIR and Ag expression, whereas low-affinity CIR was more efficient than high-affinity CIR in conditions of limiting Ag and CIR expression levels. When tumor growth inhibition was evaluated, Ag and CIR levels, rather than CIR affinity appeared relevant. Ag-driven CIR activation resulted in the production of soluble factors mediating efficient bystander effect. By carefully defining CIR surface expression and increasing affinity for a specific target antigen, it may be possible to selectively exclude CIR-mediated activity against targets expressing low levels of antigen, as normal cells. On the contrary, low antigen-expressing tumor variants could be eliminated by decreasing CIR affinity. Tuning CIR expression and affinity might help in discriminating different biologic contexts.
Journla of Immunotherapy 11/2007; 30(7):684-93. DOI:10.1097/CJI.0b013e3180de5d90 · 4.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epithelial ovarian carcinoma (EOC) remains a highly lethal malignancy. Despite the progress in surgical and therapeutic strategies, resistance to chemotherapy is still a major concern. Cytotoxic therapies mediate killing of cancer cells by activating the intrinsic mitochondrial apoptotic pathway, and p53 status is a key factor in determining the efficacy of apoptotic signaling. The extrinsic (CD95) death receptor-dependent signaling pathway also contributes to the efficacy of cancer therapy. We previously showed that EOC are generally resistant to CD95-dependent apoptosis. In p53 wild-type EOC tumors, CD95-mediated apoptosis is impaired at the receptor level by the long form of cellular FLICE-inhibitory protein, whereas this mechanism does not account for resistance in tumors with mutated p53 (p53mu). In the present study, we examined both intrinsic and death receptor-dependent apoptotic signaling in p53mu OVCAR3 EOC cell line, showing that these cells are less susceptible to cisplatin treatment as compared with p53 wild-type EOC cells and also resist CD95-mediated apoptosis due to inefficient formation of the death-inducing signaling complex and weak mitochondrial signal amplification. However, pretreatment of OVCAR3 cells with clinically relevant cisplatin concentrations significantly improved receptor-dependent apoptotic signaling by up-modulating CD95 receptor expression and increasing death-inducing signaling complex formation efficiency. The synergy of cisplatin pretreatment and CD95 triggering in inducing cell death was also shown in p53mu tumor cells derived from ascitic fluid of advanced-stage EOC patients. These findings support the effectiveness of a combined therapeutic treatment able to sensitize cancer cells to apoptosis even when p53 is functionally inactivated.
Molecular Cancer Therapeutics 03/2007; 6(2):762-72. DOI:10.1158/1535-7163.MCT-06-0357 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Based on the hypothesis that gene products involved in the same biological process would be coupled at transcriptional level, a previous study analyzed the correlation of the gene expression patterns of ligand-receptor (L-R) pairs to discover potential autocrine/paracrine signaling loops in different cancers (Graeber and Eisenberg. Nat Genet 2001; 29:295). By refining the starting database, a list of 511 L-R pairs was compiled, combined to eight data sets from a single pathology, epithelial ovarian cancer, and examined as a proof-of-principle of the statistical and biological validity of the correlation of the L-R gene expression patterns in cancer. Analysis revealed a Bonferroni-corrected significant correlation of 105 L-R pairs in at least one data set and, by systematic analysis, identified 39 more frequently correlated L-R pairs, 7 of which were already biologically confirmed. In four data sets examined for an L-R correlation associated with patient survival time, 15 L-R pairs were significantly correlated in short surviving patients in two of the data sets. Immunohistochemical analysis of one of the newly identified correlated L-R pairs (i.e., EFNB3-EPHB4) revealed the correlated expression of ephrin-B3 and EphB4 proteins in 45 of 55 epithelial ovarian tumor samples (P < 0.0001). Together, these data not only support the validity of cross-comparison analysis of gene expression data because known and expected correlations were confirmed but also point to the promise of such analysis in identifying new L-R signaling loops in cancer.
Cancer Research 12/2006; 66(22):10709-19. DOI:10.1158/0008-5472.CAN-06-1327 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two chimeric monoclonal antibodies (mAb), cMOV18 and cMOV19, recognizing distinct epitopes of folate receptor highly expressed on epithelial ovarian cancer (EOC) cells were analyzed for their ability to activate complement (C) as a means to enhance their antitumor activity. The individual cMOVs failed to activate C on six EOC cell lines as documented by the marginal deposition of C components and the negligible C-dependent cytotoxicity (CDC). Conversely, the mixture of cMOVs was more effective, although the percentage of cell killing did not exceed 25%. Fluorescence-activated cell sorting analysis of EOC cells for surface expression of the membrane C regulatory proteins (mCRP) revealed high levels of CD46, variable expression of CD59, and absence of CD55. This finding was confirmed in tumor tissue specimens obtained from advanced-stage EOC patients and analyzed for the expression of mCRPs mRNA using a cDNA microarray and for the presence of proteins by immunohistochemistry. Incubation of EOC cells with neutralizing mAbs to CD46 and CD59 led to a significant increase in the CDC from 10%-20% to 45%-50%. The relative contribution of antibody-dependent cell cytoxicity (ADCC) and C-dependent killing of two EOC cell lines induced by the mixture of cMOV18 and cMOV19 was about 15% and 25%-35%, respectively, bringing the total killing to about 40%-50%. This value increased to 60%-70% after neutralization of CD46 and CD59 without an appreciable change of ADCC. These results suggest that C is the major contributor to the killing of EOC cells induced by the mixture of cMOV18 and cMOV19.
Cancer Research 05/2006; 66(7):3876-83. DOI:10.1158/0008-5472.CAN-05-3434 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent characterization of abnormal phosphatidylcholine metabolism in tumor cells by nuclear magnetic resonance (NMR) has identified novel fingerprints of tumor progression that are potentially useful as clinical diagnostic indicators. In the present study, we analyzed the concentrations of phosphatidylcholine metabolites, activities of phosphocholine-producing enzymes, and uptake of [methyl-14C]choline in human epithelial ovarian carcinoma cell lines (EOC) compared with normal or immortalized ovary epithelial cells (EONT). Quantification of phosphatidylcholine metabolites contributing to the 1H NMR total choline resonance (3.20-3.24 ppm) revealed intracellular [phosphocholine] and [total choline] of 2.3 +/- 0.9 and 5.2 +/- 2.4 nmol/10(6) cells, respectively, with a glycerophosphocholine/phosphocholine ratio of 0.95 +/- 0.93 in EONT cells; average [phosphocholine] was 3- to 8-fold higher in EOC cells (P < 0.0001), becoming the predominant phosphatidylcholine metabolite, whereas average glycerophosphocholine/phosphocholine values decreased significantly to < or =0.2. Two-dimensional (phosphocholine/total choline, [total choline]) and (glycerophosphocholine/total choline, [total choline]) maps allowed separate clustering of EOC from EONT cells (P < 0.0001, 95% confidence limits). Rates of choline kinase activity in EOC cells were 12- to 24-fold higher (P < 0.03) than those in EONT cells (basal rate, 0.5 +/- 0.1 nmol/10(6) cells/h), accounting for a consistently elevated (5- to 15-fold) [methyl-14C]choline uptake after 1-hour incubation (P < 0.0001). The overall activity of phosphatidylcholine-specific phospholipase C and phospholipase D was also higher ( approximately 5-fold) in EOC cells, suggesting that both biosynthetic and catabolic pathways of the phosphatidylcholine cycle likely contribute to phosphocholine accumulation. Evidence of abnormal phosphatidylcholine metabolism might have implications in EOC biology and might provide an avenue to the development of noninvasive clinical tools for EOC diagnosis and treatment follow-up.
Cancer Research 11/2005; 65(20):9369-76. DOI:10.1158/0008-5472.CAN-05-1146 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Novel antibody-based immunotherapeutic strategies exploit chimeric immune receptors (CIR), expressed on the surface of transduced human peripheral blood mononuclear cells (PBMC), to redirect potent non-MHC-dependent cytotoxicity to tumor cells expressing a tumor-associated antigen. However, clinical application of the strategy has been hampered by the potential side effects associated with immunogenicity and by low transduction efficiency.
A fully human CIR was constructed that triggers immune activation through the zeta chain of CD3 and contains a human single-chain antibody fragment specific for an extracellular epitope of HER2. PBMC were transduced with the CIR using gibbon-ape leukemia virus envelope pseudotyped retroviruses. In vitro cytotoxicity and inhibition assays were carried out using normal and tumor cell lines expressing different levels of HER2.
Bulk populations of CIR-transduced PBMC could express high levels of the construct and subcloning ensured stable expression. CIR-mediated killing and growth inhibition of targets expressing high HER2 levels were very efficient at low effector-to-target ratios. Under the same experimental conditions, CIR-mediated activity against normal cells expressing low HER2 levels was marginal. The CIR-mediated recognition of target cells induced the release of soluble factors able to inhibit growth of both HER-positive and HER2-negative bystander tumor cells.
Human CIR-transduced PBMC exert a potent and dose-dependent anti-tumor activity. Target antigen level appeared to be a critical determinant of specificity and delivery of signals leading to redirected effector functions. Soluble factors, released by redirected effectors at the site of antigen-driven activation, mediate potent bystander killing.
The Journal of Gene Medicine 02/2005; 7(2):158-70. DOI:10.1002/jgm.647 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ovarian carcinoma is a highly lethal malignancy that often becomes resistant to chemotherapy. Alterations in apoptotic signals and p53 status contribute to drug resistance, and CD95-mediated apoptosis is also deficient in resistant cells. We analyzed the mechanism of resistance to CD95-mediated apoptosis in ovarian carcinoma cell lines differing in p53 status.
CD95-mediated apoptosis was induced by agonistic anti-CD95 antibody, and the apoptotic cascade was monitored with biochemical and functional assays.
CD95-mediated apoptosis was blocked in human ovarian cancer cells. In cell lines with wild-type p53, treatment with the protein synthesis inhibitor cycloheximide (CHX) together with anti-CD95 overcame the resistance, suggesting the presence of a labile inhibiting protein. Indeed, the labile protein cellular FLICE-inhibitory protein long form (c-FLIP(L)) was found to block caspase-8 recruitment to the death-inducing signaling complex (DISC), and sensitization of cells by CHX was due to c-FLIP(L) down-modulation at the DISC level. Down-regulation of c-FLIP(L) with antisense oligonucleotides increased CD95-mediated apoptosis as in cells sensitized by CHX, demonstrating the direct involvement of c-FLIP(L) in apoptosis resistance. Removal of c-FLIP(L) block at DISC level allowed full activation of the mitochondrial pathway and, eventually, apoptosis in wild-type p53 cells, whereas in cells with mutated p53, c-FLIP(L) involvement in CD95-mediated apoptosis resistance appeared to be irrelevant. Immunohistochemical analysis of an ovarian tumor tissue array revealed c-FLIP(L) expression in samples with no p53 accumulation (P = 0.034), and a significant (P = 0.037) inverse relationship between c-FLIP(L) and p53 expression levels was also observed in 27 epithelial ovarian cancer specimens with known p53 status.
The inhibitory protein c-FLIP(L) is involved in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild-type p53.
Clinical Cancer Research 09/2004; 10(15):5202-14. DOI:10.1158/1078-0432.CCR-03-0537 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aberrant glycosylation is one of the most constant traits of malignant cells. The CaMBr1 hexasaccharide antigen, originally defined on the human breast carcinoma cell line MCF7, is expressed on some normal tissues but overexpressed in a high percentage of human breast, ovary, prostate and lung carcinomas. CaMBr1 overexpression is associated with poor prognosis. The epitope consists of the tetrasaccharide Fuc(alpha1-2)Ga1(beta1-3)GalNAc(beta1-3)Ga1alpha-O-spacer, which has recently become available as a synthetic oligosaccharide. Here we report the CaMBr1 tetrasaccharide conjugation to two different carrier proteins (CRM197 and KLH) and the evaluation of conjugate immunogenicity in mice following their administration in various vaccine formulations with two adjuvants (MPL-SE and Detox-PC). Radioimmunoassay to determine the level and isotype of anti-tetrasaccharide antibodies in mouse sera, and cytofluorimetric analysis and 51Cr-release assay on human tumor cells, to evaluate specificity of binding and complement-dependent lysis respectively, identified CaMBr1-CRM197, in association with the MPL-SE adjuvant, as the best vaccine formulation. This combination induced (1) production of tetrasaccharide-specific antibodies, with negligible side-effects; (2) antibodies with complement-mediated cytotoxic activity on human CaMBr1-positive cells and (3) a high titer of IgG1 detected in sera obtained 3 months after the first injection, indicating that the anti-tetrasaccharide antibody response was mediated by T cell activation. The availability of CaMBr1-glycoconjugate in the minimal and functional antigenic structure and the identification of an efficacious vaccine formulation opens the way to exploring the activity of this glycoconjugate in a clinical setting.
Cancer Immunology and Immunotherapy 09/2000; 49(6):296-304. DOI:10.1007/s002620000113 · 3.94 Impact Factor